HER2 antibody composition

ABSTRACT

A composition comprising a main species HER2 antibody that binds to domain II of HER2, and an amino acid sequence variant thereof comprising an amino-terminal leader extension is disclosed. Pharmaceutical formulations comprising the composition, and therapeutic uses for the composition are also disclosed.

This is a divisional application which claims priority under 35 USC §120to non-provisional application Ser. No. 11/182,908 filed Jul. 15, 2005(now U.S. Pat. No. 7,560,111), which claims priority under 35 USC §119to provisional application 60/590,202 filed Jul. 22, 2004, the contentsof which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention concerns a composition comprising a main speciesHER2 antibody that binds to domain II of HER2, and an amino acidsequence variant thereof comprising an amino-terminal leader extension.The invention also relates to pharmaceutical formulations comprising thecomposition, and therapeutic uses for the composition.

BACKGROUND OF THE INVENTION HER2 Antibodies

The HER family of receptor tyrosine kinases are important mediators ofcell growth, differentiation and survival. The receptor family includesfour distinct members including epidermal growth factor receptor (EGFR,ErbB1, or HER1), HER2 (ErbB2 or p185^(neu)), HER3 (ErbB3) and HER4(ErbB4 or tyro2).

EGFR, encoded by the erbB1 gene, has been causally implicated in humanmalignancy. In particular, increased expression of EGFR has beenobserved in breast, bladder, lung, head, neck and stomach cancer as wellas glioblastomas. Increased EGFR receptor expression is often associatedwith increased production of the EGFR ligand, transforming growth factoralpha (TGF-α), by the same tumor cells resulting in receptor activationby an autocrine stimulatory pathway. Baselga and Mendelsohn Pharmac.Ther. 64:127-154 (1994). Monoclonal antibodies directed against the EGFRor its ligands, TGF-α and EGF, have been evaluated as therapeutic agentsin the treatment of such malignancies. See, e.g., Baselga andMendelsohn, supra; Masui et al. Cancer Research 44:1002-1007 (1984); andWu et al. J. Clin. Invest. 95:1897-1905 (1995).

The second member of the HER family, p185^(neu), was originallyidentified as the product of the transforming gene from neuroblastomasof chemically treated rats. The activated form of the neu proto-oncogeneresults from a point mutation (valine to glutamic acid) in thetransmembrane region of the encoded protein. Amplification of the humanhomolog of neu is observed in breast and ovarian cancers and correlateswith a poor prognosis (Slamon et al., Science, 235:177-182 (1987);Slamon et al., Science, 244:707-712 (1989); and U.S. Pat. No.4,968,603). To date, no point mutation analogous to that in the neuproto-oncogene has been reported for human tumors. Overexpression ofHER2 (frequently but not uniformly due to gene amplification) has alsobeen observed in other carcinomas including carcinomas of the stomach,endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas andbladder. See, among others, King et al., Science, 229:974 (1985); Yokotaet al., Lancet: 1:765-767 (1986); Fukushige et al., Mol Cell Biol.,6:955-958 (1986); Guerin et al., Oncogene Res., 3:21-31 (1988); Cohen etal., Oncogene, 4:81-88 (1989); Yonemura et al., Cancer Res., 51:1034(1991); Borst et al., Gynecol. Oncol., 38:364 (1990); Weiner et al.,Cancer Res., 50:421-425 (1990); Kern et al., Cancer Res., 50:5184(1990); Park et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol.Carcinog., 3:254-257 (1990); Aasland et al. Br. J. Cancer 57:358-363(1988); Williams et al. Pathobiology 59:46-52 (1991); and McCann et al.,Cancer, 65:88-92 (1990). HER2 may be overexpressed in prostate cancer(Gu et al. Cancer Lett. 99:185-9 (1996); Ross et al. Hum. Pathol.28:827-33 (1997); Ross et al. Cancer 79:2162-70 (1997); and Sadasivan etal. J. Urol. 150:126-31 (1993)).

Antibodies directed against the rat p185^(neu) and human HER2 proteinproducts have been described. Drebin and colleagues have raisedantibodies against the rat neu gene product, p185^(neu) See, forexample, Drebin et al., Cell 41:695-706 (1985); Myers et al., Meth.Enzym. 198:277-290 (1991); and WO94/22478. Drebin et al. Oncogene2:273-277 (1988) report that mixtures of antibodies reactive with twodistinct regions of p185^(neu) result in synergistic anti-tumor effectson neu-transformed NIH-3T3 cells implanted into nude mice. See also U.S.Pat. No. 5,824,311 issued Oct. 20, 1998.

Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989) describe thegeneration of a panel of HER2 antibodies which were characterized usingthe human breast tumor cell line SK-BR-3. Relative cell proliferation ofthe SK-BR-3 cells following exposure to the antibodies was determined bycrystal violet staining of the monolayers after 72 hours. Using thisassay, maximum inhibition was obtained with the antibody called 4D5which inhibited cellular proliferation by 56%. Other antibodies in thepanel reduced cellular proliferation to a lesser extent in this assay.The antibody 4D5 was further found to sensitize HER2-overexpressingbreast tumor cell lines to the cytotoxic effects of TNF-α. See also U.S.Pat. No. 5,677,171 issued Oct. 14, 1997. The HER2 antibodies discussedin Hudziak et al. are further characterized in Fendly et al. CancerResearch 50:1550-1558 (1990); Kotts et al. In Vitro 26(3):59A (1990);Sarup et al. Growth Regulation 1:72-82 (1991); Shepard et al. J. Clin.Immunol. 11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol.11(2):979-986 (1991); Lewis et al. Cancer Immunol. Immunother.37:255-263 (1993); Pietras et al. Oncogene 9:1829-1838 (1994); Vitettaet al. Cancer Research 54:5301-5309 (1994); Sliwkowski et al. J. Biol.Chem. 269(20):14661-14665 (1994); Scott et al. J. Biol. Chem.266:14300-5 (1991); D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206(1994); Lewis et al. Cancer Research 56:1457-1465 (1996); and Schaeferet al. Oncogene 15:1385-1394 (1997).

A recombinant humanized version of the murine HER2 antibody 4D5(huMAb4D5-8, rhuMAb HER2, Trastuzumab or HERCEPTIN®; U.S. Pat. No.5,821,337) is clinically active in patients with HER2-overexpressingmetastatic breast cancers that have received extensive prior anti-cancertherapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). Trastuzumabreceived marketing approval from the Food and Drug Administration Sep.25, 1998 for the treatment of patients with metastatic breast cancerwhose tumors overexpress the HER2 protein.

Other HER2 antibodies with various properties have been described inTagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al.Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991);Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski etal. PNAS (USA) 88:8691-8695 (1991); Bacus et al. Cancer Research52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993);WO94/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancocket al. Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res.54:1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994);Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No.5,783,186; and Klapper et al. Oncogene 14:2099-2109 (1997).

Homology screening has resulted in the identification of two other HERreceptor family members; HER3 (U.S. Pat. Nos. 5,183,884 and 5,480,968 aswell as Kraus et al. PNAS (USA) 86:9193-9197 (1989)) and HER4 (EP PatAppln No 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA,90:1746-1750 (1993); and Plowman et al., Nature, 366:473-475 (1993)).Both of these receptors display increased expression on at least somebreast cancer cell lines.

The HER receptors are generally found in various combinations in cellsand heterodimerization is thought to increase the diversity of cellularresponses to a variety of HER ligands (Earp et al. Breast CancerResearch and Treatment 35: 115-132 (1995)). EGFR is bound by sixdifferent ligands; epidermal growth factor (EGF), transforming growthfactor alpha (TGF-α), amphiregulin, heparin binding epidermal growthfactor (HB-EGF), betacellulin and epiregulin (Groenen et al. GrowthFactors 11:235-257 (1994)). A family of heregulin proteins resultingfrom alternative splicing of a single gene are ligands for HER3 andHER4. The heregulin family includes alpha, beta and gamma heregulins(Holmes et al., Science, 256:1205-1210 (1992); U.S. Pat. No. 5,641,869;and Schaefer et al. Oncogene 15:1385-1394 (1997)); neu differentiationfactors (NDFs), glial growth factors (GGFs); acetylcholine receptorinducing activity (ARIA); and sensory and motor neuron derived factor(SMDF). For a review, see Groenen et al. Growth Factors 11:235-257(1994); Lemke, G. Molec. & Cell. Neurosci. 7:247-262 (1996) and Lee etal. Pharm. Rev. 47:51-85 (1995). Recently three additional HER ligandswere identified; neuregulin-2 (NRG-2) which is reported to bind eitherHER3 or HER4 (Chang et al. Nature 387 509-512 (1997); and Carraway et alNature 387:512-516 (1997)); neuregulin-3 which binds HER4 (Zhang et al.PNAS (USA) 94(18):9562-7 (1997)); and neuregulin-4 which binds HER4(Harari et al. Oncogene 18:2681-89 (1999)) HB-EGF, betacellulin andepiregulin also bind to HER4.

While EGF and TGFα do not bind HER2, EGF stimulates EGFR and HER2 toform a heterodimer, which activates EGFR and results intransphosphorylation of HER2 in the heterodimer. Dimerization and/ortransphosphorylation appears to activate the HER2 tyrosine kinase. SeeEarp et al., supra. Likewise, when HER3 is co-expressed with HER2, anactive signaling complex is formed and antibodies directed against HER2are capable of disrupting this complex (Sliwkowski et al., J. Biol.Chem., 269(20):14661-14665 (1994)). Additionally, the affinity of HER3for heregulin (HRG) is increased to a higher affinity state whenco-expressed with HER2. See also, Levi et al., Journal of Neuroscience15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92:1431-1435 (1995); and Lewis et al., Cancer Res., 56:1457-1465 (1996)with respect to the HER2-HER3 protein complex. HER4, like HER3, forms anactive signaling complex with HER2 (Carraway and Cantley, Cell 78:5-8(1994)).

To target the HER signaling pathway, rhuMAb 2C4 (Pertuzumab, OMNITARG™)was developed as a humanized antibody that inhibits the dimerization ofHER2 with other HER receptors, thereby inhibiting ligand-drivenphosphorylation and activation, and downstream activation of the RAS andAKT pathways. In a phase I trial of Pertuzumab as a single agent fortreating solid tumors, 3 subjects with advanced ovarian cancer weretreated with pertuzumab. One had a durable partial response, and anadditional subject had stable disease for 15 weeks. Agus et al. Proc AmSoc Clin Oncol 22: 192, Abstract 771 (2003).

Antibody Variant Compositions

U.S. Pat. No. 6,339,142 describes a HER2 antibody composition comprisinga mixture of anti-HER2 antibody and one or more acidic variants thereof,wherein the amount of the acidic variant(s) is less than about 25%.Trastuzumab is the exemplified HER2 antibody.

Reid et al. Poster presented at Well Characterized BiotechPharmaceuticals conference (January, 2003) “Effects of Cell CultureProcess Changes on Humanized Antibody Characteristics” describes anunnamed, humanized IgG1 antibody composition with N-terminalheterogeneities due to combinations of VHS signal peptide, N-terminalglutamine, and pyroglutamic acid on the heavy chain thereof.

Reed et al. “The Ideal Chromatographic Antibody Characterization Method”Poster presented at the IBC Antibody Production Conference (February,2002) reports a VHS extension on the heavy chain of E25, a humanizedanti-IgE antibody.

Rouse et al. Poster presented at WCBP “‘Top Down’ GlycoproteinCharacterization by High Resolution Mass Spectrometry and ItsApplication to Biopharmaceutical Development” (Jan. 6-9, 2004) describesa monoclonal antibody composition with N-terminal heterogeneityresulting from ⁻³AHS or ⁻²HS signal peptide residues on the light chainthereof.

In a presentation at IBC Meeting (September, 2000) “Strategic Use ofComparability Studies and Assays for Well Characterized Biologicals,”Jill Porter discussed a late-eluting form of ZENAPAX™ with three extraamino acid residues on the heavy chain thereof.

SUMMARY OF THE INVENTION

The present invention concerns a composition comprising a main speciesHER2 antibody that binds to domain II of HER2, and an amino acidsequence variant thereof comprising an amino-terminal leader extension.

In addition, the invention provides a composition comprising a mixtureof a main species HER2 antibody comprising variable light and variableheavy sequences in SEQ ID Nos. 3 and 4, respectively, and an amino acidsequence variant of the main species antibody comprising aVHS-amino-terminal leader extension attached to one or two variablelight domains thereof, wherein from about 1% to about 20% of antibodymolecules in the composition comprise a VHS-amino-terminal leaderextension.

The invention further concerns a polypeptide comprising the amino acidsequence in SEQ ID No. 23, or a deamidated and/or oxidizedvariant-thereof, as well as an antibody comprising (a) a light chaincomprising that polypeptide, and (b) a heavy chain comprising an aminoacid sequence selected from the group consisting of SEQ ID NO. 16, SEQID NO. 24, and a deamidated and/or oxidized variant of SEQ ID NO. 16 orSEQ ID NO. 24.

The invention also concerns a pharmaceutical formulation comprising thecomposition in a pharmaceutically acceptable carrier, and a method oftreating HER2-expressing cancer in a patient comprising administeringthe pharmaceutical formulation to the patient in an amount effective totreat the cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic of the HER2 protein structure, and aminoacid sequences for Domains I-IV (SEQ ID Nos. 19-22, respectively) of theextracellular domain thereof.

FIGS. 2A and 2B depict alignments of the amino acid sequences of thevariable light (V_(L)) (FIG. 2A) and variable heavy (V_(H)) (FIG. 2B)domains of murine monoclonal antibody 2C4 (SEQ ID Nos. 1 and 2,respectively); V_(L) and V_(H) domains of humanized 2C4 version 574 (SEQID Nos. 3 and 4, respectively), and human V_(L) and V_(H) consensusframeworks (hum κ1, light kappa subgroup I; humIII, heavy subgroup III)(SEQ ID Nos. 5 and 6, respectively). Asterisks identify differencesbetween humanized 2C4 version 574 and murine monoclonal antibody 2C4 orbetween humanized 2C4 version 574 and the human framework.Complementarity Determining Regions (CDRs) are in brackets.

FIGS. 3A and 3B show the amino acid sequences of Pertuzumab light chain(SEQ ID No. 15) and heavy chain (SEQ ID No. 16). CDRs are shown in bold.The carbohydrate moiety is attached to Asn 299 of the heavy chain.

FIGS. 4A and 4B show the amino acid sequences of Pertuzumab light chain(SEQ ID No. 17) and heavy chain, each including an intact amino terminalsignal peptide sequence (SEQ ID No. 18).

FIG. 5 depicts, schematically, binding of 2C4 at the heterodimericbinding site of HER2, thereby preventing heterodimerization withactivated EGFR or HER3.

FIG. 6 depicts coupling of HER2/HER3 to the MAPK and Akt pathways.

FIG. 7 compares activities of Trastuzumab and Pertuzumab.

FIGS. 8A-1, 8A-2, 8A-3, 8B-1, 8B-2 and 8B-3 show reconstructed massspectra of reduced Pertuzumab light chain (FIGS. 8A-1, 8A-2 and 8A-3)and heavy chain (FIGS. 8B-1, 8B-2 and 8B-3).

FIGS. 9A and 9B depict cation exchange chromatography analysis of nativePertuzumab (FIG. 9A) and CPB-digested Pertuzumab (FIG. 9B).

FIG. 10 shows size exclusion chromatographic analysis of Pertuzumab.

FIGS. 11A and 11B show CE-SDS-LIF analysis of reduced Pertuzumab (FIG.11A) and intact Pertuzumab (FIG. 11B).

FIGS. 12A and 12B depict tryptic peptide maps of Pertuzumab (FIG. 12A),and LYS-C peptide maps of Pertuzumab (FIG. 12B).

FIG. 13 shows CE analysis of N-linked oligosaccharides released fromPertuzumab.

FIGS. 14A and 14B show oligosaccharide structures commonly observed inIgG antibodies.

FIG. 15 depicts positive mode MALDI-TOF mass spectra of neutraloligosaccharides released from Pertuzumab.

FIGS. 16A and 16B show the amino acid sequences of Trastuzumab lightchain (SEQ ID No. 13) and heavy chain (SEQ ID No. 14).

FIGS. 17A and 17B depict a variant Pertuzumab light chain sequence (SEQID No. 23) and a variant Pertuzumab heavy chain sequence (SEQ ID No.24).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The term “main species antibody” herein refers to the antibody aminoacid sequence structure in a composition which is the quantitativelypredominant antibody molecule in the composition. Preferably, the mainspecies antibody is a HER2 antibody, such as an antibody that binds toDomain II of HER2, antibody that inhibits HER dimerization moreeffectively than Trastuzumab, and/or an antibody which binds to aheterodimeric binding site of HER2. The preferred embodiment herein ofthe main species antibody is one comprising the variable light andvariable heavy amino acid sequences in SEQ ID Nos. 3 and 4, and mostpreferably comprising the light chain and heavy chain amino acidsequences in SEQ ID Nos. 15 and 16 (Pertuzumab).

An “amino acid sequence variant” antibody herein is an antibody with anamino acid sequence which differs from a main species antibody.Ordinarily, amino acid sequence variants will possess at least about 70%homology with the main species antibody, and preferably, they will be atleast about 80%, and more preferably at least about 90% homologous withthe main species antibody. The amino acid sequence variants possesssubstitutions, deletions, and/or additions at certain positions withinor adjacent to the amino acid sequence of the main species antibody.Examples of amino acid sequence variants herein include an acidicvariant (e.g. a deamidated antibody variant), a basic variant, theantibody with an amino-terminal leader extension (e.g. VHS-) on one ortwo light chains thereof, antibody with a C-terminal lysine residue onone or two heavy chains thereof, antibody with one or more oxidizedmethionine residues, etc. and includes combinations of variations to theamino acid sequences of heavy and/or light chains. The antibody variantof particular interest herein is the antibody comprising anamino-terminal leader extension on one or two light chains thereof,optionally further comprising other amino acid sequence and/orglycosylation differences relative to the main species antibody.

A “glycosylation variant” antibody herein is an antibody with one ormore carbohydrate moeities attached thereto which differ from one ormore carbohydrate moieties attached to a main species antibody. Examplesof glycosylation variants herein include antibody with a G1 or G2oligosaccharide structure, instead a G0 oligosaccharide structure,attached to an Fc region thereof, antibody with one or two carbohydratemoieties attached to one or two light chains thereof, antibody with nocarbohydrate attached to one or two heavy chains of the antibody, etc,as well as combinations of such glycosylation alterations.

Where the antibody has an Fc region, an oligosaccharide structure suchas that shown in FIG. 14 herein may be attached to one or two heavychains of the antibody, e.g. at residue 299. For Pertuzumab, G0 was thepredominant oligosaccharide structure, with other oligosaccharidestructures such as G0-F, G-1, Man5, Man6, G1-1, G1(1-6), G1(1-3) and G2being found in lesser amounts in the Pertuzumab composition.

Unless indicated otherwise, a “G1 oligosaccharide structure” hereinincludes G1(1-6) and G1(1-3) structures.

An “amino-terminal leader extension” herein refers to one or more aminoacid residues of the amino-terminal leader sequence that are present atthe amino-terminus of any one or more heavy or light chains of anantibody. An exemplary amino-terminal leader extension comprises orconsists of three amino acid residues, VHS, present on one or both lightchains of an antibody variant.

A “deamidated” antibody is one in which one or more asparagine residuesthereof has been derivitized, e.g. to an aspartic acid, a succinimide,or an iso-aspartic acid.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. One such computer program is “Align 2”, authored by Genentech,Inc., which was filed with user documentation in the United StatesCopyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

For the purposes herein, “cation exchange analysis” refers to any methodby which a composition comprising two or more compounds is separatedbased on charge differences using a cation exchanger. A cation exchangergenerally comprises covalently bound, negatively charged groups.Preferably, the cation exchanger herein is a weak cation-exchangerand/or comprises a carboxylate functional group, such as the PROPACWCX-10™ cation exchange column sold by Dionex.

A “HER receptor” is a receptor protein tyrosine kinase which belongs tothe HER receptor family and includes EGFR, HER2, HER3 and HER4 receptorsand other members of this family to be identified in the future. The HERreceptor will generally comprise an extracellular domain, which may bindan HER ligand; a lipophilic transmembrane domain; a conservedintracellular tyrosine kinase domain; and a carboxyl-terminal signalingdomain harboring several tyrosine residues which can be phosphorylated.Preferably the HER receptor is native sequence human HER receptor.

The extracellular domain of HER2 comprises four domains, Domain I (aminoacid residues from about 1-195), Domain II (amino acid residues fromabout 196-319), Domain III (amino acid residues from about 320-488), andDomain IV (amino acid residues from about 489-630) (residue numberingwithout signal peptide). See Garrett et al. Mol. Cell. 11: 495-505(2003), Cho et al. Nature 421: 756-760 (2003), Franklin et al. CancerCell 5:317-328 (2004), or Plowman et al. Proc. Natl. Acad. Sci.90:1746-1750 (1993). See, also, FIG. 1 herein.

The terms “ErbB1,” “HER1”, “epidermal growth factor receptor” and “EGFR”are used interchangeably herein and refer to EGFR as disclosed, forexample, in Carpenter et al. Ann. Rev. Biochem. 56:881-914 (1987),including naturally occurring mutant forms thereof (e.g. a deletionmutant EGFR as in Humphrey et al. PNAS (USA) 87:4207-4211 (1990)). erbB1refers to the gene encoding the EGFR protein product.

The expressions “ErbB2” and “HER2” are used interchangeably herein andrefer to human HER2 protein described, for example, in Semba et al.,PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234(1986) (Genebank accession number X03363). The term “erbB2” refers tothe gene encoding human ErbB2 and “neu” refers to the gene encoding ratp185^(neu). Preferred HER2 is native sequence human HER2.

“ErbB3” and “HER3” refer to the receptor polypeptide as disclosed, forexample, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus etal. PNAS (USA) 86:9193-9197 (1989).

The terms “ErbB4” and “HER4” herein refer to the receptor polypeptide asdisclosed, for example, in EP Pat Appln No 599,274; Plowman et al.,Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al.,Nature, 366:473-475 (1993), including isoforms thereof, e.g., asdisclosed in WO99/19488, published Apr. 22, 1999.

By “HER ligand” is meant a polypeptide which binds to and/or activatesan HER receptor. The HER ligand of particular interest herein is anative sequence human HER ligand such as epidermal growth factor (EGF)(Savage et al., J. Biol. Chem. 247:7612-7621 (1972)); transforminggrowth factor alpha (TGF-α) (Marquardt et al., Science 223:1079-1082(1984)); amphiregulin also known as schwanoma or keratinocyte autocrinegrowth factor (Shoyab et al. Science 243:1074-1076 (1989); Kimura et al.Nature 348:257-260 (1990); and Cook et al. Mol. Cell. Biol. 11:2547-2557(1991)); betacellulin (Shing et al., Science 259:1604-1607 (1993); andSasada et al. Biochem. Biophys. Res. Commun. 190:1173 (1993));heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al.,Science 251:936-939 (1991)); epiregulin (Toyoda et al., J. Biol. Chem.270:7495-7500 (1995); and Komurasaki et al. Oncogene 15:2841-2848(1997)); a heregulin (see below); neuregulin-2 (NRG-2) (Carraway et al.,Nature 387:512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al., Proc.Natl. Acad. Sci. 94:9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari etal. Oncogene 18:2681-89 (1999)) or cripto (CR-1) (Kannan et al. J. Biol.Chem. 272(6):3330-3335 (1997)). HER ligands which bind EGFR include EGF,TGF-α, amphiregulin, betacellulin, HB-EGF and epiregulin. HER ligandswhich bind HER3 include heregulins. HER ligands capable of binding HER4include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4 andheregulins.

“Heregulin” (HRG) when used herein refers to a polypeptide encoded bythe heregulin gene product as disclosed in U.S. Pat. No. 5,641,869 orMarchionni et al., Nature, 362:312-318 (1993). Examples of heregulinsinclude heregulin-α, heregulin-β1, heregulin-β2 and heregulin-β3 (Holmeset al., Science, 256:1205-1210 (1992); and U.S. Pat. No. 5,641,869); neudifferentiation factor (NDF) (Peles et al. Cell 69: 205-216 (1992));acetylcholine receptor-inducing activity (ARIA) (Falls et al. Cell72:801-815 (1993)); glial growth factors (GGFs) (Marchionni et al.,Nature, 362:312-318 (1993)); sensory and motor neuron derived factor(SMDF) (Ho et al. J. Biol. Chem. 270:14523-14532 (1995)); γ-heregulin(Schaefer et al. Oncogene 15:1385-1394 (1997)). The term includesbiologically active fragments and/or amino acid sequence variants of anative sequence HRG polypeptide, such as an EGF-like domain fragmentthereof (e.g. HRGβ1₁₇₇₋₂₄₄).

A “HER dimer” herein is a noncovalently associated dimer comprising atleast two different HER receptors. Such complexes may form when a cellexpressing two or more HER receptors is exposed to an HER ligand and canbe isolated by immunoprecipitation and analyzed by SDS-PAGE as describedin Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994), forexample. Examples of such HER dimers include EGFR-HER2, HER2-HER3 andHER3-HER4 heterodimers. Moreover, the HER dimer may comprise two or moreHER2 receptors combined with a different HER receptor, such as HER3,HER4 or EGFR. Other proteins, such as a cytokine receptor subunit (e.g.gp130) may be associated with the dimer.

A “heterodimeric binding site” on HER2, refers to a region in theextracellular domain of HER2 that contacts, or interfaces with, a regionin the extracellular domain of EGFR, HER3 or HER4 upon formation of adimer therewith. The region is found in Domain II of HER2. Franklin etal. Cancer Cell 5:317-328 (2004).

“HER activation” or “HER2 activation” refers to activation, orphosphorylation, of any one or more HER receptors, or HER2 receptors.Generally, HER activation results in signal transduction (e.g. thatcaused by an intracellular kinase domain of a HER receptorphosphorylating tyrosine residues in the HER receptor or a substratepolypeptide). HER activation may be mediated by HER ligand binding to aHER dimer comprising the HER receptor of interest. HER ligand binding toa HER dimer may activate a kinase domain of one or more of the HERreceptors in the dimer and thereby results in phosphorylation oftyrosine residues in one or more of the HER receptors and/orphosphorylation of tyrosine residues in additional substratepolypeptides(s), such as Akt or MAPK intracellular kinases.

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments, so long as theyexhibit the desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such as thosevariants described herein. In contrast to polyclonal antibodypreparations that typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey are uncontaminated by other immunoglobulins. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al, Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimericantibodies of interest herein include “primatized” antibodies comprisingvariable domain antigen-binding sequences derived from a non-humanprimate (e.g. Old World Monkey, Ape etc) and human constant regionsequences.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibodyfragment(s).

An “intact antibody” is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H)1, C_(H)2 and C_(H)3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variants thereof. Preferably, the intactantibody has one or more effector functions, and comprises anoligosaccharide structure attached to one or two heavy chains thereof.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (e.g. B cell receptor; BCR), etc.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells in summarized is Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source thereof, e.g. from blood or PBMCs asdescribed herein.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and Fcγ RIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (seereview M. in Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g. an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163(1996), may be performed.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend. The constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light-chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994). HER2 antibody scFv fragments are described in WO93/16185; U.S.Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(V_(H)) connected to a variable light domain (V_(L)) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3,huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 orTrastuzumab (HERCEPTIN®) as described in Table 3 of U.S. Pat. No.5,821,337 expressly incorporated herein by reference; humanized 520C9(WO93/21319) and humanized 2C4 antibodies as described herein.

For the purposes herein, “Trastuzumab,” “HERCEPTIN®,” and “huMAb4D5-8”refer to an antibody comprising the light and heavy chain amino acidsequences in SEQ ID NOS. 13 and 14, respectively.

Herein, “Pertuzumab,” “OMNITARG™,” and “rhuMAb 2C4,” refer to anantibody comprising the variable light and variable heavy amino acidsequences in SEQ ID Nos. 3 and 4, respectfully. Where Pertuzumab is anintact antibody, it preferably comprises the light chain and heavy chainamino acid sequences in SEQ ID Nos. 15 and 16, respectively.

A “naked antibody” is an antibody (as herein defined) that is notconjugated to a heterologous molecule, such as a cytotoxic moiety orradiolabel.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

A HER2 antibody which “inhibits HER dimerization more effectively thanTrastuzumab” is one which reduces or eliminates HER dimers moreeffectively (for example at least about 2-fold more effectively) thanTrastuzumab. Preferably, such an antibody inhibits HER2 dimerization atleast about as effectively as an antibody selected from the groupconsisting of intact murine monoclonal antibody 2C4, a Fab fragment ofmurine monoclonal antibody 2C4, intact Pertuzumab, and a Fab fragment ofPertuzumab. One can evaluate HER dimerization inhibition by studying HERdimers directly, or by evaluating HER activation, or downstreamsignaling, which results from HER dimerization, and/or by evaluating theantibody-HER2 binding site, etc. Assays for screening for antibodieswith the ability to inhibit HER dimerization more effectively thanTrastuzumab are described in Agus et al. Cancer Cell 2: 127-137 (2002)and WO01/00245 (Adams et al.). By way of example only, one may assay forinhibition of HER dimerization by assessing, for example, inhibition ofHER dimer formation (see, e.g., FIG. 1A-B of Agus et al. Cancer Cell 2:127-137 (2002); and WO01/00245); reduction in HER ligand activation ofcells which express HER dimers (WO01/00245 and FIG. 2A-B of Agus et al.Cancer Cell 2: 127-137 (2002), for example); blocking of HER ligandbinding to cells which express HER dimers (WO01/00245, and FIG. 2E ofAgus et al. Cancer Cell 2: 127-137 (2002), for example); cell growthinhibition of cancer cells (e.g. MCF7, MDA-MD-134, ZR-75-1, MD-MB-175,T-47D cells) which express HER dimers in the presence (or absence) ofHER ligand (WO01/00245 and FIGS. 3A-D of Agus et al. Cancer Cell 2:127-137 (2002), for instance); inhibition of downstream signaling (forinstance, inhibition of HRG-dependent AKT phosphorylation or inhibitionof HRG- or TGFα-dependent MAPK phosphorylation) (see, WO01/00245, andFIG. 2C-D of Agus et al. Cancer Cell 2: 127-137 (2002), for example).One may also assess whether the antibody inhibits HER dimerization bystudying the antibody-HER2 binding site, for instance, by evaluating astructure or model, such as a crystal structure, of the antibody boundto HER2 (See, for example, Franklin et al. Cancer Cell 5:317-328(2004)).

The HER2 antibody may “inhibit HRG-dependent AKT phosphorylation” and/orinhibit “HRG- or TGFα-dependent MAPK phosphorylation” more effectively(for instance at least 2-fold more effectively) than Trastuzumab (seeAgus et al. Cancer Cell 2: 127-137 (2002) and WO01/00245, by way ofexample).

The HER2 antibody may be one which does “not inhibit HER2 ectodomaincleavage” (Molina et al. Cancer Res. 61:4744-4749 (2001)).

A HER2 antibody that “binds to a heterodimeric binding site” of HER2,binds to residues in domain II (and optionally also binds to residues inother of the domains of the HER2 extracellular domain, such as domains Iand III), and can sterically hinder, at least to some extent, formationof a HER2-EGFR, HER2-HER3, or HER2-HER4 heterodimer. Franklin et al.Cancer Cell 5:317-328 (2004) characterize the HER2-Pertuzumab crystalstructure, deposited with the RCSB Protein Data Bank (ID Code IS78),illustrating an exemplary antibody that binds to the heterodimericbinding site of HER2.

An antibody that “binds to domain II” of HER2 binds to residues indomain II and optionally residues in other domain(s) of HER2, such asdomains I and III. Preferably the antibody that binds to domain II bindsto the junction between domains I, II and III of HER2.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially a HER expressingcancer cell either in vitro or in vivo. Thus, the growth inhibitoryagent may be one which significantly reduces the percentage of HERexpressing cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topo II inhibitors such as doxorubicin,epirubicin, daunorubicin, etoposide, and bleomycin. Those agents thatarrest G1 also spill over into S-phase arrest, for example, DNAalkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13.

Examples of “growth inhibitory” antibodies are those which bind to HER2and inhibit the growth of cancer cells overexpressing HER2. Preferredgrowth inhibitory HER2 antibodies inhibit growth of SK-BR-3 breast tumorcells in cell culture by greater than 20%, and preferably greater than50% (e.g. from about 50% to about 100%) at an antibody concentration ofabout 0.5 to 30 μg/ml, where the growth inhibition is determined sixdays after exposure of the SK-BR-3 cells to the antibody (see U.S. Pat.No. 5,677,171 issued Oct. 14, 1997). The SK-BR-3 cell growth inhibitionassay is described in more detail in that patent and hereinbelow. Thepreferred growth inhibitory antibody is a humanized variant of murinemonoclonal antibody 4D5, e.g., Trastuzumab.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is usually one which overexpresses the HER2 receptor. Preferablythe cell is a tumor cell, e.g. a breast, ovarian, stomach, endometrial,salivary gland, lung, kidney, colon, thyroid, pancreatic or bladdercell. In vitro, the cell may be a SK-BR-3, BT474, Calu 3 cell,MDA-MB-453, MDA-MB-361 or SKOV3 cell. Various methods are available forevaluating the cellular events associated with apoptosis. For example,phosphatidyl serine (PS) translocation can be measured by annexinbinding; DNA fragmentation can be evaluated through DNA laddering; andnuclear/chromatin condensation along with DNA fragmentation can beevaluated by any increase in hypodiploid cells. Preferably, the antibodywhich induces apoptosis is one which results in about 2 to 50 fold,preferably about 5 to 50 fold, and most preferably about 10 to 50 fold,induction of annexin binding relative to untreated cell in an annexinbinding assay using BT474 cells (see below). Examples of HER2 antibodiesthat induce apoptosis are 7C2 and 7F3.

The “epitope 2C4” is the region in the extracellular domain of HER2 towhich the antibody 2C4 binds. In order to screen for antibodies whichbind to the 2C4 epitope, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to assess whether theantibody binds to the 2C4 epitope of HER2 using methods known in the artand/or one can study the antibody-HER2 structure (Franklin et al. CancerCell 5:317-328 (2004)) to see what domain(s) of HER2 is/are bound by theantibody. Epitope 2C4 comprises residues from domain II in theextracellular domain of HER2. 2C4 and Pertuzumab bind to theextracellular domain of HER2 at the junction of domains I, II and III.Franklin et al. Cancer Cell 5:317-328 (2004).

The “epitope 4D5” is the region in the extracellular domain of HER2 towhich the antibody 4 D5 (ATCC CRL 10463) and Trastuzumab bind. Thisepitope is close to the transmembrane domain of HER2, and within DomainIV of HER2. To screen for antibodies which bind to the 4D5 epitope, aroutine cross-blocking assay such as that described in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed. Alternatively, epitope mapping can beperformed to assess whether the antibody binds to the 4D5 epitope ofHER2 (e.g. any one or more residues in the region from about residue 529to about residue 625, inclusive, in FIG. 1).

The “epitope 7C2/7F3” is the region at the N terminus, within Domain I,of the extracellular domain of HER2 to which the 7C2 and/or 7F3antibodies (each deposited with the ATCC, see below) bind. To screen forantibodies which bind to the 7C2/7F3 epitope, a routine cross-blockingassay such as that described in Antibodies, A Laboratory Manual, ColdSpring Harbor Laboratory, Ed Harlow and David Lane (1988), can beperformed. Alternatively, epitope mapping can be performed to establishwhether the antibody binds to the 7C2/7F3 epitope on HER2 (e.g. any oneor more of residues in the region from about residue 22 to about residue53 of HER2 in FIG. 1).

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disease as well as those in which the disease is to beprevented. Hence, the patient to be treated herein may have beendiagnosed as having the disease or may be predisposed or susceptible tothe disease.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma (including medulloblastoma andretinoblastoma), sarcoma (including liposarcoma and synovial cellsarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma,and islet cell cancer), mesothelioma, schwannoma (including acousticneuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, testicular cancer,esophagael cancer, tumors of the biliary tract, as well as head and neckcancer.

The term “effective amount” refers to an amount of a drug effective totreat disease in the patient. Where the disease is cancer, the effectiveamount of the drug may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the cancer. To the extent the drug may preventgrowth and/or kill existing cancer cells, it may be cytostatic and/orcytotoxic. The effective amount may extend progression free survival,result in an objective response (including a partial response, PR, orcomplete response, CR), increase overall survival time, and/or improveone or more symptoms of cancer.

A “HER2-expressing cancer” is one comprising cells which have HER2protein present at their cell surface.

A cancer which “overexpresses” a HER receptor is one which hassignificantly higher levels of a HER receptor, such as HER2, at the cellsurface thereof, compared to a noncancerous cell of the same tissuetype. Such overexpression may be caused by gene amplification or byincreased transcription or translation. HER receptor overexpression maybe determined in a diagnostic or prognostic assay by evaluatingincreased levels of the HER protein present on the surface of a cell(e.g. via an immunohistochemistry assay; IHC). Alternatively, oradditionally, one may measure levels of HER-encoding nucleic acid in thecell, e.g. via fluorescent in situ hybridization (FISH; see WO98/45479published October, 1998), southern blotting, or polymerase chainreaction (PCR) techniques, such as real time quantitative PCR (RT-PCR).One may also study HER receptor overexpression by measuring shed antigen(e.g., HER extracellular domain) in a biological fluid such as serum(see, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995;and Sias et al. J. Immunol. Methods 132: 73-80 (1990)). Aside from theabove assays, various in vivo assays are available to the skilledpractitioner. For example, one may expose cells within the body of thepatient to an antibody which is optionally labeled with a detectablelabel, e.g. a radioactive isotope, and binding of the antibody to cellsin the patient can be evaluated, e.g. by external scanning forradioactivity or by analyzing a biopsy taken from a patient previouslyexposed to the antibody.

Conversely, a cancer which “does not overexpress HER2 receptor” is onewhich does not express higher than normal levels of HER2 receptorcompared to a noncancerous cell of the same tissue type.

A cancer which “overexpresses” a HER ligand is one which producessignificantly higher levels of that ligand compared to a noncancerouscell of the same tissue type. Such overexpression may be caused by geneamplification or by increased transcription or translation.Overexpression of the HER ligand may be determined diagnostically byevaluating levels of the ligand (or nucleic acid encoding it) in thepatient, e.g. in a tumor biopsy or by various diagnostic assays such asthe IHC, FISH, southern blotting, PCR or in vivo assays described above.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e. g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®,morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®),liposomal doxorubicin TLC D-99 (MYOCET®), peglylated liposomaldoxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate,gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), anepothilone, and 5-fluorouracil (5-FU); folic acid analogues such asdenopterin, methotrexate, pteropterin, trimetrexate; purine analogs suchas fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;anti-adrenals such as aminoglutethimide, mitotane, trilostane; folicacid replenisher such as frolinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elfornithine; elliptinium acetate; etoglucid; gallium nitrate;hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine andansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene,Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™),and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; platinum agents such as cisplatin, oxaliplatin, andcarboplatin; vincas, which prevent tubulin polymerization from formingmicrotubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®),vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®);etoposide (VP-16); ifosfamide; mitoxantrone; leucovovin; novantrone;edatrexate; daunomycin; aminopterin; ibandronate; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such asretinoic acid, including bexarotene (TARGRETIN®); bisphosphonates suchas clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®),NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®),pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g.,ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine,COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor(e.g. PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577);orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium(GENASENSE®); pixantrone; EGFR inhibitors (see definition below);tyrosine kinase inhibitors (see definition below); and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above such as CHOP, an abbreviationfor a combined therapy of cyclophosphamide, doxorubicin, vincristine,and prednisolone, and FOLFOX, an abbreviation for a treatment regimenwith oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens withmixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®),4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene,raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogenreceptor modulators (SERMs) such as SERM3; pure anti-estrogens withoutagonist properties, such as fulvestrant (FASLODEX®), and EM800 (suchagents may block estrogen receptor (ER) dimerization, inhibit DNAbinding, increase ER turnover, and/or suppress ER levels); aromataseinhibitors, including steroidal aromatase inhibitors such as formestaneand exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors suchas anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide,and other aromatase inhibitors including vorozole (RIVISOR®), megestrolacetate (MEGASE®), fadrozole, imidazole; lutenizing hormone-releasinghormone agonists, including leuprolide (LUPRON® and ELIGARD®),goserelin, buserelin, and tripterelin; sex steroids, includingprogestines such as megestrol acetate and medroxyprogesterone acetate,estrogens such as diethylstilbestrol and premarin, andandrogens/retinoids such as fluoxymesterone, all transretionic acid andfenretinide; onapristone; anti-progesterones; estrogen receptordown-regulators (ERDs); anti-androgens such as flutamide, nilutamide andbicalutamide; testolactone; and pharmaceutically acceptable salts, acidsor derivatives of any of the above; as well as combinations of two ormore of the above.

As used herein, the term “EGFR-targeted drug” refers to a therapeuticagent that binds to EGFR and, optionally, inhibits EGFR activation.Examples of such agents include antibodies and small molecules that bindto EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCCCRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.)and variants thereof, such as chimerized 225 (C225 or Cetuximab;ERBUTIX®) and reshaped human 225 (H225) (see, WO 96/40210, ImcloneSystems Inc.); IMC-11F8, a fully human, EGFR-targeted antibody(Imclone); antibodies that bind type II mutant EGFR (U.S. Pat. No.5,212,290); humanized and chimeric antibodies that bind EGFR asdescribed in U.S. Pat. No. 5,891,996; and human antibodies that bindEGFR, such as ABX-EGF or Panitumumab (see WO98/50433, Abgenix/Amgen);EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996));EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR thatcompetes with both EGF and TGF-alpha for EGFR binding (EMD/Merck); humanEGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known asE1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3 and described inU.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanizedmAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Theanti-EGFR antibody may be conjugated with a cytotoxic agent, thusgenerating an immunoconjugate (see, e.g., EP659,439A2, Merck PatentGmbH). EGFR antagonists include small molecules such as compoundsdescribed in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307,5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726,6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459,6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, aswell as the following PCT publications: WO98/14451, WO98/50038,WO99/09016, and WO99/24037. Particular small molecule EGFR antagonistsinclude OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech/OSIPharmaceuticals); PD 183805 (CI 1033, 2-propenamide,N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-,dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA™)4-(3′-Chloro-4′-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline,AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline,Zeneca); BIBX-1382(N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine,Boehringer Ingelheim); PKI-166((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol);(R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine);CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide);EKB-569(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide)(Wyeth); AG1478 (Sugen); AG1571 (SU 5271; Sugen); dual EGFR/HER2tyrosine kinase inhibitors such as lapatinib (GW 572016 orN-[3-chloro-4-[(3fluorophenyl)methoxy]phenyl]6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine;Glaxo-SmithKline) or cyanoguanidine quinazoline and cyanoamidinequinazolamine derivatives.

A “tyrosine kinase inhibitor” is a molecule which inhibits tyrosinekinase activity of a tyrosine kinase such as a HER receptor. Examples ofsuch inhibitors include the EGFR-targeted drugs noted in the precedingparagraph; small molecule HER2 tyrosine kinase inhibitor such as TAK165available from Takeda; CP-724,714, an oral selective inhibitor of theErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitorssuch as EKB-569 (available from Wyeth) which preferentially binds EGFRbut inhibits both HER2 and EGFR-overexpressing cells; lapatinib(GW572016; available from Glaxo-SmithKline) an oral HER2 and EGFRtyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HERinhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitorssuch as antisense agent ISIS-5132 available from ISIS Pharmaceuticalswhich inhibits Raf-1 signaling; non-HER targeted TK inhibitors such asImatinib mesylate (GLEEVAC™) available from Glaxo; MAPK extracellularregulated kinase I inhibitor CI-1040 (available from Pharmacia);quinazolines, such as PD 153035, 4-(3-chloroanilino) quinazoline;pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules(e.g. those that bind to HER-encoding nucleic acid); quinoxalines (U.S.Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474(Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors suchas CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinibmesylate (Gleevac; Novartis); PKI 166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen);ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11(Imclone); cyanoguanidine quinazoline and cyanoamidine quinazolaminederivatives; or as described in any of the following patentpublications: U.S. Pat. No. 5,804,396; WO99/09016 (American Cyanamid);WO98/43960 (American Cyanamid); WO97/38983 (Warner Lambert); WO99/06378(Warner Lambert); WO99/06396 (Warner Lambert); WO96/30347 (Pfizer, Inc);WO96/33978 (Zeneca); WO96/3397 (Zeneca); WO96/33980 (Zeneca); andUS2005/0101617.

An “anti-angiogenic agent” refers to a compound which blocks, orinterferes with to some degree, the development of blood vessels. Theanti-angiogenic factor may, for instance, be a small molecule orantibody that binds to a growth factor or growth factor receptorinvolved in promoting angiogenesis. The preferred anti-angiogenic factorherein is an antibody that binds to Vascular Endothelial Growth Factor(VEGF), such as Bevacizumab (AVASTIN®).

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such asTNF-α or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

II. HER2 Antibody Variant Compositions

The present invention concerns, at least in part, certain HER2 antibodycompositions. Preferably, the HER2 antibody (either or both of the mainspecies HER2 antibody and antibody variant thereof) is one which bindsto Domain II of HER2, inhibits HER dimerization more effectively thanTrastuzumab, and/or binds to a heterodimeric binding site of HER2. Thepreferred embodiment herein of the main species antibody is onecomprising the variable light and variable heavy amino acid sequences inSEQ ID Nos. 3 and 4, and most preferably comprising the light chain andheavy chain amino acid sequences in SEQ ID Nos. 15 and 16 (Pertuzumab).

The composition herein comprises a mixture of the main species HER2antibody and an amino acid sequence variant thereof comprising anamino-terminal leader extension. Preferably, the amino-terminal leaderextension is on a light chain of the antibody variant (e.g. on one ortwo light chains of the antibody variant). The main species HER2antibody or the antibody variant may be an intact antibody or antibodyfragment (e.g. Fab of F(ab′)2 fragments), but preferably both are intactantibodies.

The antibody variant herein comprises an amino-terminal leader extensionon any one or more of the heavy or light chains thereof. Preferably, theamino-terminal leader extension is on one or two light chains of theantibody. The amino-terminal leader extension preferably comprises orconsists of VHS-.

Presence of the amino-terminal leader extension in the composition canbe detected by various analytical techniques including, but not limitedto, N-terminal sequence analysis, assay for charge heterogeneity (forinstance, cation exchange chromatography or capillary zoneelectrophoresis), mass spectrometry, etc. The amount of the antibodyvariant in the composition generally ranges from an amount thatconstitutes the lower detection limit of any assay (preferably cationexchange analysis) used to detect the variant to an amount less than theamount of the main species antibody. Generally, about 20% or less (e.g.from about 1% to about 15%, for instance from 5% to about 15%, andpreferably from about 8% to about 12%) of the antibody molecules in thecomposition comprise an amino-terminal leader extension. Such percentageamounts are preferably determined using cation exchange analysis.

Aside from the amino-terminal leader extension variant, further aminoacid sequence alterations of the main species antibody and/or variantare contemplated, including but not limited to an antibody comprising aC-terminal lysine residue on one or both heavy chains thereof (such anantibody variant may be present in an amount from about 1% to about20%), a deamidated antibody variant (for instance, wherein Asn-386and/or Asn-391 on one or two heavy chains of Pertuzumab are deamidated),antibody with one or more oxidized methionine residues (for example,Pertuzumab comprising oxidized met-254) etc.

Moreover, the main species antibody or variant may further compriseglycosylation variations, non-limiting examples of which includeantibody comprising a G1 or G2 oligosaccharide structure attached to theFc region thereof, antibody comprising a carbohydrate moiety attached toa light chain thereof (e.g. one or two carbohydrate moieties, such asglucose or galactose, attached to one or two light chains of theantibody, for instance attached to one or more lysine residues),antibody comprising one or two non-glycosylated heavy chains, orantibody comprising a sialidated oligosaccharide attached to one or twoheavy chains thereof etc.

The invention also concerns a polypeptide comprising the amino acidsequence in SEQ ID No. 23 or a deamidated and/or oxidized variantthereof. In addition, the invention provides an antibody comprising oneor two light chains, wherein either or both of the light chains comprisethe amino acid sequence in SEQ ID No. 23. The antibody further comprisesone or two heavy chains, wherein either or both of the heavy chainscomprise the amino acid sequence in SEQ ID NO. 16 or SEQ ID NO. 24 (ordeamidated and/or oxidized variants thereof).

The composition may be recovered from a genetically engineered cellline, e.g. a Chinese Hamster Ovary (CHO) cell line expressing the HER2antibody, or may be prepared by peptide synthesis.

III. Production of HER2 Antibodies

A description follows as to exemplary techniques for the production ofthe antibodies used in accordance with the present invention. The HER2antigen to be used for production of antibodies may be, e.g., a solubleform of the extracellular domain of HER2 or a portion thereof,containing the desired epitope. Alternatively, cells expressing HER2 attheir cell surface (e.g. NIH-3T3 cells transformed to overexpress HER2;or a carcinoma cell line such as SK-BR-3 cells, see Stancovski et al.PNAS (USA) 88:8691-8695 (1991)) can be used to generate antibodies.Other forms of HER2 useful for generating antibodies will be apparent tothose skilled in the art.

(i) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(ii) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical and/or bind the same epitope, except forpossible variants that may arise during production of the monoclonalantibody, such as those variants described herein. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al, Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al, Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al, Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al, Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy chain and light chain constant domains in placeof the homologous murine sequences (U.S. Pat. No. 4,816,567; andMorrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iii) Humanized Antibodies

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting hypervariable region sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol, 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

WO01/00245 describes production of exemplary humanized HER2 antibodieswhich bind HER2 and block ligand activation of a HER receptor. Thehumanized antibody of particular interest herein blocks EGF, TGF-αand/or HRG mediated activation of MAPK essentially as effectively asintact murine monoclonal antibody 2C4 (or a Fab fragment thereof) and/orbinds HER2 essentially as effectively as intact murine monoclonalantibody 2C4 (or a Fab fragment thereof). The humanized antibody hereinmay, for example, comprise nonhuman hypervariable region residuesincorporated into a human variable heavy domain and may further comprisea framework region (FR) substitution at a position selected from thegroup consisting of 69H, 71H and 73H utilizing the variable domainnumbering system set forth in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991). In one embodiment, thehumanized antibody comprises FR substitutions at two or all of positions69H, 71H and 73H.

An exemplary humanized antibody of interest herein comprises variableheavy complementarity determining residues GFTFTDYTMX, where X ispreferably D or S (SEQ ID NO:7); DVNPNSGGSIYNQRFKG (SEQ ID NO:8); and/orNLGPSFYFDY (SEQ ID NO:9), optionally comprising amino acid modificationsof those CDR residues, e.g. where the modifications essentially maintainor improve affinity of the antibody. For example, the antibody variantof interest may have from about one to about seven or about five aminoacid substitutions in the above variable heavy CDR sequences. Suchantibody variants may be prepared by affinity maturation, e.g., asdescribed below. The most preferred humanized antibody comprises thevariable heavy amino acid sequence in SEQ ID NO:4.

The humanized antibody may comprise variable light complementaritydetermining residues KASQDVSIGVA (SEQ ID NO: 10); SASYX¹X²X³, where X¹is preferably R or L, X² is preferably Y or E, and X³ is preferably T orS (SEQ ID NO:1); and/or QQYYIYPYT (SEQ ID NO:12), e.g. in addition tothose variable heavy domain CDR residues in the preceding paragraph.Such humanized antibodies optionally comprise amino acid modificationsof the above CDR residues, e.g. where the modifications essentiallymaintain or improve affinity of the antibody. For example, the antibodyvariant of interest may have from about one to about seven or about fiveamino acid substitutions in the above variable light CDR sequences. Suchantibody variants may be prepared by affinity maturation, e.g., asdescribed below. The most preferred humanized antibody comprises thevariable light amino acid sequence in SEQ ID NO:3.

The present application also contemplates affinity matured antibodieswhich bind HER2 and block ligand activation of a HER receptor. Theparent antibody may be a human antibody or a humanized antibody, e.g.,one comprising the variable light and/or variable heavy sequences of SEQID Nos. 3 and 4, respectively (i.e. variant 574). The affinity maturedantibody preferably binds to HER2 receptor with an affinity superior tothat of intact murine 2C4 or intact variant 574 (e.g. from about two orabout four fold, to about 100 fold or about 1000 fold improved affinity,e.g. as assessed using a HER2-extracellular domain (ECD) ELISA).Exemplary variable heavy CDR residues for substitution include H28, H30,H34, H35, H64, H96, H99, or combinations of two or more (e.g. two,three, four, five, six, or seven of these residues). Examples ofvariable light CDR residues for alteration include L28, L50, L53, L56,L91, L92, L93, L94, L96, L97 or combinations of two or more (e.g. two tothree, four, five or up to about ten of these residues).

Various forms of the humanized antibody or affinity matured antibody arecontemplated. For example, the humanized antibody or affinity maturedantibody may be an antibody fragment, such as a Fab, which is optionallyconjugated with one or more cytotoxic agent(s) in order to generate animmunoconjugate. Alternatively, the humanized antibody or affinitymatured antibody may be an intact antibody, such as an intact IgG1antibody.

(iv) Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669,5,589,369 and 5,545,807.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human HER2 antibodies are described in U.S. Pat. No. 5,772,997 issuedJun. 30, 1998 and WO 97/00271 published Jan. 3, 1997.

(v) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. Theantibody fragment may also be a “linear antibody”, e.g., as described inU.S. Pat. No. 5,641,870 for example. Such linear antibody fragments maybe monospecific or bispecific.

(vi) Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the HER2 protein. Other suchantibodies may combine a HER2 binding site with binding site(s) forEGFR, HER3 and/or HER4. Alternatively, a HER2 arm may be combined withan arm which binds to a triggering molecule on a leukocyte such as aT-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG(FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as tofocus cellular defense mechanisms to the HER2-expressing cell.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express HER2. These antibodies possess a HER2-binding armand an arm which binds the cytotoxic agent (e.g. saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

WO 96/16673 describes a bispecific HER2/FcγRIII antibody and U.S. Pat.No. 5,837,234 discloses a bispecific HER2/FcγRI antibody IDM1 (Osidem).A bispecific HER2/Fcα antibody is shown in WO98/02463. U.S. Pat. No.5,821,337 teaches a bispecific HER2/CD3 antibody. MDX-210 is abispecific HER2-FcγRIII Ab.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

(vii) Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the HER2 antibodies describedherein are contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the HER2 antibody are prepared byintroducing appropriate nucleotide changes into the HER2 antibodynucleic acid, or by peptide synthesis. Such modifications include, forexample, deletions from, and/or insertions into and/or substitutions of,residues within the amino acid sequences of the HER2 antibody. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the HER2 antibody, such as changing thenumber or position of glycosylation sites.

A useful method for identification of certain residues or regions of theHER2 antibody that are preferred locations for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and WellsScience, 244:1081-1085 (1989). Here, a residue or group of targetresidues are identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with HER2 antigen. Those amino acid locationsdemonstrating functional sensitivity to the substitutions then arerefined by introducing further or other variants at, or for, the sitesof substitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed HER2 antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includea HER2 antibody with an N-terminal methionyl residue or the antibodyfused to a cytotoxic polypeptide. Other insertional variants of the HER2antibody molecule include the fusion to the N- or C-terminus of the HER2antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increasesthe serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the HER2 antibodymolecule replaced by a different residue. The sites of greatest interestfor substitutional mutagenesis include the hypervariable regions orCDRs, but FR or Fc region alterations are also contemplated.Conservative substitutions are shown in Table 1 under the heading of“preferred substitutions”. If such substitutions result in a change inbiological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75,Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (Q)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the HER2 antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g. 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and huma HER2. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites),

Where the antibody comprises an Fc region, any oligosaccharide structureattached thereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 A1, Presta, L.See also US 2004/0093621 A1 (Kyowa Hakko Kogyo Co., Ltd). Antibodieswith a bisecting N-acetylglucosamine (GlcNAc) in the oligosaccharidestructure attached to an Fc region of the antibody are referenced inWO03/011878, Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana etal. Antibodies with at least one galactose residue in an oligosaccharidestructure attached to an Fc region of the antibody are reported inWO97/30087, Patel et al. See, also, WO98/58964 (Raju, S.) and WO99/22764(Raju, S.) concerning antibodies with altered carbohydrate attached tothe Fc region thereof. Antibody compositions comprising main speciesantibody with such carbohydrate structures attached to one or two heavychains of the Fc region are contemplated herein.

Nucleic acid molecules encoding amino acid sequence variants of the HER2antibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the HER2 antibody.

(viii) Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above. One mayfurther select antibodies with certain biological characteristics, asdesired.

To identify an antibody which blocks ligand activation of a HERreceptor, the ability of the antibody to block HER ligand binding tocells expressing the HER receptor (e.g. in conjugation with another HERreceptor with which the HER receptor of interest forms a HERhetero-oligomer) may be determined. For example, cells naturallyexpressing, or transfected to express, HER receptors of the HERhetero-oligomer may be incubated with the antibody and then exposed tolabeled HER ligand. The ability of the HER2 antibody to block ligandbinding to the HER receptor in the HER hetero-oligomer may then beevaluated.

For example, inhibition of HRG binding to MCF7 breast tumor cell linesby HER2 antibodies may be performed using monolayer MCF7 cultures on icein a 24-well-plate format essentially as described in WO 01/00245. HER2monoclonal antibodies may be added to each well and incubated for 30minutes. ¹²⁵I-labeled rHRGβ1₁₇₇₋₂₂₄ (25 pm) may then be added, and theincubation may be continued for 4 to 16 hours. Dose response curves maybe prepared and an IC₅₀ value may be calculated for the antibody ofinterest. In one embodiment, the antibody which blocks ligand activationof an HER receptor will have an IC₅₀ for inhibiting HRG binding to MCF7cells in this assay of about 50 nM or less, more preferably 10 nM orless. Where the antibody is an antibody fragment such as a Fab fragment,the IC₅₀ for inhibiting HRG binding to MCF7 cells in this assay may, forexample, be about 100 nM or less, more preferably 50 nM or less.

Alternatively, or additionally, the ability of the HER2 antibody toblock HER ligand-stimulated tyrosine phosphorylation of a HER receptorpresent in a HER hetero-oligomer may be assessed. For example, cellsendogenously expressing the HER receptors or transfected to expressedthem may be incubated with the antibody and then assayed for HERligand-dependent tyrosine phosphorylation activity using ananti-phosphotyrosine monoclonal (which is optionally conjugated with adetectable label). The kinase receptor activation assay described inU.S. Pat. No. 5,766,863 is also available for determining HER receptoractivation and blocking of that activity by an antibody.

In one embodiment, one may screen for an antibody which inhibits HRGstimulation of p180 tyrosine phosphorylation in MCF7 cells essentiallyas described in WO01/00245. For example, the MCF7 cells may be plated in24-well plates and monoclonal antibodies to HER2 may be added to eachwell and incubated for 30 minutes at room temperature; thenrHRGβ1₁₇₇₋₂₄₄ may be added to each well to a final concentration of 0.2nM, and the incubation may be continued for 8 minutes. Media may beaspirated from each well, and reactions may be stopped by the additionof 100 μl of SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl,pH 6.8). Each sample (25 μl) may be electrophoresed on a 4-12% gradientgel (Novex) and then electrophoretically transferred to polyvinylidenedifluoride membrane. Antiphosphotyrosine (at 1 μg/ml) immunoblots may bedeveloped, and the intensity of the predominant reactive band atM_(r)˜180,000 may be quantified by reflectance densitometry. Theantibody selected will preferably significantly inhibit HRG stimulationof p180 tyrosine phosphorylation to about 0-35% of control in thisassay. A dose-response curve for inhibition of HRG stimulation of p180tyrosine phosphorylation as determined by reflectance densitometry maybe prepared and an IC₅₀ for the antibody of interest may be calculated.In one embodiment, the antibody which blocks ligand activation of a HERreceptor will have an IC₅₀ for inhibiting HRG stimulation of p180tyrosine phosphorylation in this assay of about 50 nM or less, morepreferably 10 nM or less. Where the antibody is an antibody fragmentsuch as a Fab fragment, the IC₅₀ for inhibiting HRG stimulation of p180tyrosine phosphorylation in this assay may, for example, be about 100 nMor less, more preferably 50 nM or less.

One may also assess the growth inhibitory effects of the antibody onMDA-MB-175 cells, e.g, essentially as described in Schaefer et al.Oncogene 15:1385-1394 (1997). According to this assay, MDA-MB-175 cellsmay treated with a HER2 monoclonal antibody (10 μg/mL) for 4 days andstained with crystal violet. Incubation with a HER2 antibody may show agrowth inhibitory effect on this cell line similar to that displayed bymonoclonal antibody 2C4. In a further embodiment, exogenous HRG will notsignificantly reverse this inhibition. Preferably, the antibody will beable to inhibit cell proliferation of MDA-MB-175 cells to a greaterextent than monoclonal antibody 4D5 (and optionally to a greater extentthan monoclonal antibody 7F3), both in the presence and absence ofexogenous HRG.

In one embodiment, the HER2 antibody of interest may block heregulindependent association of HER2 with HER3 in both MCF7 and SK-BR-3 cellsas determined in a co-immunoprecipitation experiment such as thatdescribed in WO01/00245 substantially more effectively than monoclonalantibody 4D5, and preferably substantially more effectively thanmonoclonal antibody 7F3.

To identify growth inhibitory HER2 antibodies, one may screen forantibodies which inhibit the growth of cancer cells which overexpressHER2. In one embodiment, the growth inhibitory antibody of choice isable to inhibit growth of SK-BR-3 cells in cell culture by about 20-100%and preferably by about 50-100% at an antibody concentration of about0.5 to 30 μg/ml. To identify such antibodies, the SK-BR-3 assaydescribed in U.S. Pat. No. 5,677,171 can be performed. According to thisassay, SK-BR-3 cells are grown in a 1:1 mixture of F12 and DMEM mediumsupplemented with 10% fetal bovine serum, glutamine and penicillinstreptomycin. The SK-BR-3 cells are plated at 20,000 cells in a 35 mmcell culture dish (2 mls/35 mm dish). 0.5 to 30 μg/ml of the HER2antibody is added per dish. After six days, the number of cells,compared to untreated cells are counted using an electronic COULTER™cell counter. Those antibodies which inhibit growth of the SK-BR-3 cellsby about 20-100% or about 50-100% may be selected as growth inhibitoryantibodies. See U.S. Pat. No. 5,677,171 for assays for screening forgrowth inhibitory antibodies, such as 4D5 and 3E8.

In order to select for antibodies which induce apoptosis, an annexinbinding assay using BT474 cells is available. The BT474 cells arecultured and seeded in dishes as discussed in the preceding paragraph.The medium is then removed and replaced with fresh medium alone ormedium containing 10 μg/ml of the monoclonal antibody. Following a threeday incubation period, monolayers are washed with PBS and detached bytrypsinization. Cells are then centrifuged, resuspended in Ca²⁺ bindingbuffer and aliquoted into tubes as discussed above for the cell deathassay. Tubes then receive labeled annexin (e.g. annexin V-FTIC) (1μg/ml). Samples may be analyzed using a FACSCAN™ flow cytometer andFACSCONVERT™ CellQuest software (Becton Dickinson). Those antibodieswhich induce statistically significant levels of annexin bindingrelative to control are selected as apoptosis-inducing antibodies. Inaddition to the annexin binding assay, a DNA staining assay using BT474cells is available. In order to perform this assay, BT474 cells whichhave been treated with the antibody of interest as described in thepreceding two paragraphs are incubated with 9 μg/ml HOECHST 33342™ for 2hr at 37° C., then analyzed on an EPICS ELITE™ flow cytometer (CoulterCorporation) using MODFIT LT™ software (Verity Software House).Antibodies which induce a change in the percentage of apoptotic cellswhich is 2 fold or greater (and preferably 3 fold or greater) thanuntreated cells (up to 100% apoptotic cells) may be selected aspro-apoptotic antibodies using this assay. See WO98/17797 for assays forscreening for antibodies which induce apoptosis, such as 7C2 and 7F3.

To screen for antibodies which bind to an epitope on HER2 bound by anantibody of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed to assesswhether the antibody cross-blocks binding of an antibody, such as 2C4 orPertuzumab, to HER2. Alternatively, or additionally, epitope mapping canbe performed by methods known in the art and/or one can study theantibody-HER2 structure (Franklin et al. Cancer Cell 5:317-328 (2004))to see what domain(s) of HER2 is/are bound by the antibody.

(ix) Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g. a small molecule toxin or an enzymatically active toxin ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof), or a radioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Conjugates of an antibodyand one or more small molecule toxins, such as a calicheamicin, amaytansine (U.S. Pat. No. 5,208,020), a trichothene, and CC1065 are alsocontemplated herein.

In one preferred embodiment of the invention, the antibody is conjugatedto one or more maytansine molecules (e.g. about 1 to about 10 maytansinemolecules per antibody molecule). Maytansine may, for example, beconverted to May-SS-Me which may be reduced to May-SH3 and reacted withmodified antibody (Chari et al. Cancer Research 52: 127-131 (1992)) togenerate a maytansinoid-antibody immunoconjugate.

Another immunoconjugate of interest comprises a HER2 antibody conjugatedto one or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. Structural analogues of calicheamicinwhich may be used include, but are not limited to, γ₁ ^(I), α₂ ^(I), α₃^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I) ₁ (Hinman et al. Cancer Research53: 3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928(1998)). See, also, U.S. Pat. Nos. 5,714,586; 5,712,374; 5,264,586; and5,773,001 expressly incorporated herein by reference.

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g. aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

A variety of radioactive isotopes are available for the production ofradioconjugated HER2 antibodies. Examples include At²¹¹, I¹³¹, I¹²⁵,Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, dimethyl linker or disulfide-containinglinker (Chari et al. Cancer Research 52: 127-131 (1992)) may be used.

Alternatively, a fusion protein comprising the HER2 antibody andcytotoxic agent may be made, e.g. by recombinant techniques or peptidesynthesis.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g. avidin) whichis conjugated to a cytotoxic agent (e.g. a radionucleotide).

(x) Other Antibody Modifications

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol. The antibody also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980).

It may be desirable to modify the antibody of the invention with respectto effector function, e.g. so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al. Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

WO00/42072 (Presta, L.) describes antibodies with improved ADCC functionin the presence of human effector cells, where the antibodies compriseamino acid substitutions in the Fc region thereof. Preferably, theantibody with improved ADCC comprises substitutions at positions 298,333, and/or 334 of the Fc region. Preferably the altered Fc region is ahuman IgG1 Fc region comprising or consisting of substitutions at one,two or three of these positions.

Antibodies with altered C1q binding and/or complement dependentcytotoxicity (CDC) are described in WO99/51642, U.S. Pat. No.6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No. 6,528,624B1 andU.S. Pat. No. 6,538,124 (Idusogie et al.). The antibodies comprise anamino acid substitution at one or more of amino acid positions 270, 322,326, 327, 329, 313, 333 and/or 334 of the Fc region thereof.

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule. Antibodies with substitutions in an Fc region thereofand increased serum half-lives are also described in WO00/42072 (Presta,L.).

Engineered antibodies with three or more (preferably four) functionalantigen binding sites are also contemplated (US Appln No. US2002/0004587A1, Miller et al.).

The HER2 antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; andWO97/38731 published Oct. 23, 1997. Liposomes with enhanced circulationtime are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al. J. National Cancer Inst. 81 (19) 1484 (1989).

IV. Pharmaceutical Formulations

Therapeutic formulations of the compositions of the present inventionare prepared for storage by mixing the composition with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Lyophilized HER2 antibody formulations are described in WO 97/04801.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide antibodies whichbind to EGFR, HER2 (e.g. an antibody which binds a different epitope onHER2), HER3, HER4, or vascular endothelial factor (VEGF) in the oneformulation. Alternatively, or additionally, the composition may furthercomprise a chemotherapeutic agent, cytotoxic agent, cytokine, growthinhibitory agent, anti-hormonal agent, EGFR-targeted drug,anti-angiogenic agent, tyrosine kinase inhibitor, and/orcardioprotectant. Such molecules are suitably present in combination inamounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

V. Screening Patients for Therapy

According to a preferred embodiment of the invention herein, the patientselected for therapy has a tumor, or other cell or tissue, displayingHER (and preferably HER2) activation. In one embodiment, the extent ofHER (or HER2) activation in cancer cells or other cells testedsignificantly exceeds the level of activation of that receptor innon-cancerous or normal cells of the same tissue type. Such excessiveactivation may result from overexpression of the HER receptor and/orgreater than normal levels of a HER ligand available for activating theHER receptor in the cancer cells. Such excessive activation may causeand/or be caused by the malignant state of a cancer cell. In someembodiments, the cancer will be subjected to a diagnostic or prognosticassay to determine whether amplification and/or overexpression of a HERreceptor is occurring which results in such excessive activation of theHER receptor. Alternatively, or additionally, the cancer may besubjected to a diagnostic or prognostic assay to determine whetheramplification and/or overexpression a HER ligand is occurring in thecancer which contributes to excessive activation of the receptor. In asubset of such cancers, excessive activation of the receptor may resultfrom an autocrine stimulatory pathway. Various exemplary assays fordetermining HER activation will be described in more detail below.

(i) HER Dimers

Samples can be assessed for the presence of HER dimers, as indicatingHER or HER2 activation. Any method known in the art may be used todetect HER2 dimers, such as EGFR-HER2, HER2-HER3. Several preferredmethods are described below. These methods detect noncovalentprotein-protein interactions or otherwise indicate proximity betweenproteins of interest.

Immunoaffinity-based methods, such as immunoprecipitation or ELISA, maybe used to detect HER dimers. In one embodiment, HER2 antibodies areused to immunoprecipitate complexes comprising HER2 from tumor cells,and the resulting immunoprecipitant is then probed for the presence ofEGFR or HER3 by immunoblotting. In another embodiment, EGFR or HER3antibodies may be used for the immunoprecipitation step and theimmunoprecipitant then probed with HER2 antibodies. In a furtherembodiment, HER ligands specific to EGFR, HER3, EGFR-HER2 complexes orHER2-HER3 complexes may be used to precipitate complexes, which are thenprobed for the presence of HER2. For example, ligands may be conjugatedto avidin and complexes purified on a biotin column.

In other embodiments, such as ELISA or antibody “sandwich”-type assays,antibodies to HER2 are immobilized on a solid support, contacted withtumor cells or tumor cell lysate, washed, and then exposed to antibodyagainst EGFR or HER3. Binding of the latter antibody, which may bedetected directly or by a secondary antibody conjugated to a detectablelabel, indicates the presence of heterodimers. In certain embodiments,EGFR or HER3 antibody is immobilized, and HER2 antibody is used for thedetection step. In other embodiments HER ligands may be used in placeof, or in combination with HER antibodies.

Chemical or UV cross-linking may also be used to covalently join dimerson the surface of living cells. Hunter et al., Biochem. J., 320:847-53.Examples of chemical cross-linkers include dithiobis(succinimidyl)propionate (DSP) and 3,3′dithiobis(sulphosuccinimidyl) propionate(DTSSP). In one embodiment, cell extracts from chemically cross-linkedtumor cells are analyzed by SDS-PAGE and immunoblotted with antibodiesto EGFR and/or HER3. A supershifted band of the appropriate molecularweight most likely represents EGFR-HER2 or HER2-HER3 dimers, as HER2 isthe preferred dimerization partner for EGFR and HER3. This result may beconfirmed by subsequent immunoblotting with HER2 antibodies.

Fluorescence resonance energy transfer (FRET) may also be used to detectEGFR-HER2 or HER2-HER3 dimers. FRET detects protein conformationalchanges and protein-protein interactions in vivo and in vitro based onthe transfer of energy from a donor fluorophore to an acceptorfluorophore. Selvin, Nat. Struct. Biol., 7:730-34 (2000). Energytransfer takes place only if the donor fluorophore is in sufficientproximity to the acceptor fluorophore. In a typical FRET experiment, twoproteins or two sites on a single protein are labeled with differentfluorescent probes. One of the probes, the donor probe, is excited to ahigher energy state by incident light of a specified wavelength. Thedonor probe then transmits its energy to the second probe, the acceptorprobe, resulting in a reduction in the donor's fluorescence intensityand an increase in the acceptor's fluorescence emission. To measure theextent of energy transfer, the donor's intensity in a sample labeledwith donor and acceptor probes is compared with its intensity in asample labeled with donor probe only. Optionally, acceptor intensity iscompared in donor/acceptor and acceptor only samples. Suitable probesare known in the art and include, for example, membrane permeant dyes,such as fluorescein and rhodamine, organic dyes, such as the cyaninedyes, and lanthanide atoms. Selvin, supra. Methods and instrumentationfor detecting and measuring energy transfer are also known in the art.Selvin, supra.

FRET-based techniques suitable for detecting and measuringprotein-protein interactions in individual cells are also known in theart. For example, donor photobleaching fluorescence resonance energytransfer (pbFRET) microscopy and fluorescence lifetime imagingmicroscopy (FLIM) may be used to detect the dimerization of cell surfacereceptors. Selvin, supra; Gadella & Jovin, J. Cell Biol., 129:1543-58(1995). In one embodiment, pbFRET is used on cells either “insuspension” or “in situ” to detect and measure the formation ofEGFR-HER2 or HER2-HER3 dimers, as described in Nagy et al., Cytometry,32:120-131 (1998). These techniques measure the reduction in a donor'sfluorescence lifetime due to energy transfer. In a particularembodiment, a flow cytometric Foerster-type FRET technique (FCET) may beused to investigate EGFR-HER2 and HER2-HER3 dimerization, as describedin Nagy et al., supra, and Brockhoff et al., Cytometry, 44:338-48(2001).

FRET is preferably used in conjunction with standard immunohistochemicallabeling techniques. Kenworthy, Methods, 24:289-96 (2001). For example,antibodies conjugated to suitable fluorescent dyes can be used as probesfor labeling two different proteins. If the proteins are withinproximity of one another, the fluorescent dyes act as donors andacceptors for FRET. Energy transfer is detected by standard means.Energy transfer may be detected by flow cytometric means or by digitalmicroscopy systems, such as confocal microscopy or wide-fieldfluorescence microscopy coupled to a charge-coupled device (CCD) camera.

In one embodiment of the present invention, HER2 antibodies and eitherEGFR or HER3 antibodies are directly labeled with two differentfluorophores, for example as described in Nagy et al, supra. Tumor cellsor tumor cell lysates are contacted with the differentially labeledantibodies, which act as donors and acceptors for FRET in the presenceof EGFR-HER2 or HER2-HER3 dimers. Alternatively, unlabeled antibodiesagainst HER2 and either EGFR or HER3 are used along with differentiallylabeled secondary antibodies that serve as donors and acceptors. See,for example, Brockhoff et al., supra. Energy transfer is detected andthe presence of dimers is determined if the labels are found to be inclose proximity.

In other embodiments HER receptor ligands that are specific for HER2 andeither EGFR or HER3 are fluorescently labeled and used for FRET studies.

In still other embodiments of the present invention, the presence ofdimers on the surface of tumor cells is demonstrated by co-localizationof HER2 with either EGFR or HER3 using standard direct or indirectimmunofluorescence techniques and confocal laser scanning microscopy.Alternatively, laser scanning imaging (LSI) is used to detect antibodybinding and co-localization of HER2 with either EGFR or HER3 in ahigh-throughput format, such as a microwell plate, as described in Zucket al, Proc. Natl. Acad. Sci. USA, 96:11122-27 (1999).

In further embodiments, the presence of EGFR-HER2 and/or HER2-HER3dimers is determined by identifying enzymatic activity that is dependentupon the proximity of the dimer components. A HER2 antibody isconjugated with one enzyme and an EGFR or HER3 antibody is conjugatedwith a second enzyme. A first substrate for the first enzyme is addedand the reaction produces a second substrate for the second enzyme. Thisleads to a reaction with another molecule to produce a detectablecompound, such as a dye. The presence of another chemical breaks downthe second substrate, so that reaction with the second enzyme isprevented unless the first and second enzymes, and thus the twoantibodies, are in close proximity. In a particular embodiment tumorcells or cell lysates are contacted with a HER2 antibody that isconjugated with glucose oxidase and a HER3 or HER1 antibody that isconjugated with horse radish peroxidase. Glucose is added to thereaction, along with a dye precursor, such as DAB, and catalase. Thepresence of dimers is determined by the development of color uponstaining for DAB.

Dimers may also be detected using methods based on the eTag™ assaysystem (Aclara Bio Sciences, Mountain View, Calif.), as described, forexample, in U.S. Patent Application 2001/0049105, published Dec. 6,2001, both of which are expressly incorporated by reference in theirentirety. An eTag™, or “electrophoretic tag,” comprises a detectablereporter moiety, such as a fluorescent group. It may also comprise a“mobility modifier,” which consists essentially of a moiety having aunique electrophoretic mobility. These moieties allow for separation anddetection of the eTag™ from a complex mixture under definedelectrophoretic conditions, such as capillary electrophoresis (CE). Theportion of the eTag™ containing the reporter moiety and, optionally, themobility modifier is linked to a first target binding moiety by acleavable linking group to produce a first binding compound. The firsttarget binding moiety specifically recognizes a particular first target,such as a nucleic acid or protein. The first target binding moiety isnot limited in any way, and may be for example, a polynucleotide or apolypeptide. Preferably, the first target binding moiety is an antibodyor antibody fragment. Alternatively, the first target binding moiety maybe a HER receptor ligand or binding-competent fragment thereof.

The linking group preferably comprises a cleavable moiety, such as anenzyme substrate, or any chemical bond that may be cleaved under definedconditions. When the first target binding moiety binds to its target,the cleaving agent is introduced and/or activated, and the linking groupis cleaved, thus releasing the portion of the eTag™ containing thereporter moiety and mobility modifier. Thus, the presence of a “free”eTag™ indicates the binding of the target binding moiety to its target.

Preferably, a second binding compound comprises the cleaving agent and asecond target binding moiety that specifically recognizes a secondtarget. The second target binding moiety is also not limited in any wayand may be, for example, an antibody or antibody fragment or a HERreceptor ligand or binding competent ligand fragment. The cleaving agentis such that it will only cleave the linking group in the first bindingcompound if the first binding compound and the second binding compoundare in close proximity.

In an embodiment of the present invention, a first binding compoundcomprises an eTag™ in which an antibody to HER2 serves as the firsttarget binding moiety. A second binding compound comprises an antibodyto EGFR or HER3 joined to a cleaving agent capable of cleaving thelinking group of the eTag™. Preferably the cleaving agent must beactivated in order to be able to cleave the linking group. Tumor cellsor tumor cell lysates are contacted with the eTag™, which binds to HER2,and with the modified EGFR or HER3 antibody, which binds to EGFR or HER3on the cell surface. Unbound binding compound is preferable removed, andthe cleaving agent is activated, if necessary. If EGFR-HER2 or HER2-HER3dimers are present, the cleaving agent will cleave the linking group andrelease the eTag™ due to the proximity of the cleaving agent to thelinking group. Free eTag™ may then be detected by any method known inthe art, such as capillary electrophoresis.

In one embodiment, the cleaving agent is an activatable chemical speciesthat acts on the linking group. For example, the cleaving agent may beactivated by exposing the sample to light.

In another embodiment, the eTag™ is constructed using an antibody toEGFR or HER3 as the first target binding moiety, and the second bindingcompound is constructed from an antibody to HER2.

In yet another embodiment, the HER dimer is detected using an antibodyor other reagent which specifically or preferentially binds to the dimeras compared to binding thereof to either HER receptor in the dimer.

(ii) HER2 Phosphorylation

Immunoprecipitation with EGFR, HER2, or HER3 antibody as discussed inthe previous section may optionally be followed by a functional assayfor dimers, as an alternative or supplement to immunoblotting. In oneembodiment, immunoprecipitation with HER3 antibody is followed by anassay for receptor tyrosine kinase activity in the immunoprecipitant.Because HER3 does not have intrinsic tyrosine kinase activity, thepresence of tyrosine kinase activity in the immunoprecipitant indicatesthat HER3 is most likely associated with HER2. Graus-Porta et al., EMBOJ., 16:1647-55 (1997); Klapper et al., Proc. Natl. Acad. Sci. USA,96:4995-5000 (1999). This result may be confirmed by immunoblotting withHER2 antibodies. In another embodiment, immunoprecipitation with HER2antibody is followed by an assay for EGFR receptor tyrosine kinaseactivity. In this assay, the immunoprecipitant is contacted withradioactive ATP and a peptide substrate that mimics the in vivo site oftransphosphorylation of HER2 by EGFR. Phosphorylation of the peptideindicates co-immunoprecipitation and thus dimerization of EGFR withHER2. Receptor tyrosine kinase activity assays are well known in the artand include assays that detect phosphorylation of target substrates, forexample, by phosphotyrosine antibody, and activation of cognate signaltransduction pathways, such as the MAPK pathway.

Phosphorylation of HER receptor may be assessed by immunoprecipitationof one or more HER receptors, such as HER2 (HER2) receptor, and Westernblot analysis. For example, positivity is determined by the presence ofa phospho-HER2 band on the gel, using an anti-phosphotyrosine antibodyto detect phosphorylated tyrosine residue(s) in the immunoprecipitatedHER receptor(s). Anti-phosphotyrosine antibodies are commerciallyavailable from PanVera (Madison, Wis.), a subsidiary of Invitrogen,Chemicon International Inc. (Temecula, Calif.), or Upstate Biotechnology(Lake Placid, N.Y.). Negativity is determined by the absence of theband.

In another embodiment, phosphorylation of HER2 (HER2) receptor isassessed by immunohistochemistry using a phospho-specific HER2 antibody(clone PN2A; Thor et al., J. Clin. Oncol., 18(18):3230-3239 (2000)).

Other methods for detecting phosphorylation of HER receptor(s) include,but are not limited to, KIRA ELISA (U.S. Pat. Nos. 5,766,863; 5,891,650;5,914,237; 6,025,145; and 6,287,784), mass spectrometry (comparing sizeof phosphorylated and non-phosphorylated HER2), and e-tag proximityassay with both a HER (e.g. HER2) antibody and phospho-specific orphospho-tyrosine specific antibody (e.g., using the eTag™ assay kitavailable from Aclara BioSciences (Mountain View, Calif.). Details ofthe eTag assay are described hereinabove.

One may also use phospho-specific antibodies in cellular array to detectphosphorylation status in a cellular sample of signal transductionprotein (US 2003/0190689).

(iii) HER2 Ligands

Levels of a HER ligand, such as TGF-α, in or associated with the tumormay be determined according to known procedures. Such assays may detectprotein and/or nucleic acid encoding it in the sample to be tested. Inone embodiment, HER ligand levels in the tumor may be determined usingimmunohistochemistry (IHC); see, for example, Scher et al. Clin. CancerResearch 1:545-550 (1995). Alternatively, or additionally, one mayevaluate levels of HER ligand-encoding nucleic acid in the sample to betested; e.g. via FISH, southern blotting, or PCR techniques.

(iv) Non-HER2 Overexpressing Cancer

While the cancer may be characterized by overexpression of the HER2receptor, the present application further provides a method for treatingcancer which is not considered to be a HER2-overexpressing.

To determine HER2 expression in the cancer, variousdiagnostic/prognostic assays are available. In one embodiment, HER2overexpression may be analyzed by IHC, e.g. using the HERCEPTEST®(Dako). Parrafin embedded tissue sections from a tumor biopsy may besubjected to the IHC assay and accorded a HER2 protein stainingintensity criteria as follows:

Score 0 no staining is observed or membrane staining is observed in lessthan 10% of tumor cells.

Score 1+ a faint/barely perceptible membrane staining is detected inmore than 10% of the tumor cells. The cells are only stained in part oftheir membrane.

Score 2+ a weak to moderate complete membrane staining is observed inmore than 10% of the tumor cells.

Score 3+ a moderate to strong complete membrane staining is observed inmore than 10% of the tumor cells.

Those tumors with 0 or 1+ scores for HER2 overexpression assessment maybe characterized as not overexpressing HER2, whereas those tumors with2+ or 3+ scores may be characterized as overexpressing HER2.

Tumors overexpressing HER2 may be rated by immunohistochemical scorescorresponding to the number of copies of HER2 molecules expressed percell, and can been determined biochemically:

0=0-10,000 copies/cell,

1+=at least about 200,000 copies/cell,

2+=at least about 500,000 copies/cell,

3+=at least about 2,000,000 copies/cell.

Overexpression of HER2 at the 3+ level, which leads toligand-independent activation of the tyrosine kinase (Hudziak et al.,Proc. Natl. Acad. Sci. USA, 84:7159-7163 (1987)), occurs inapproximately 30% of breast cancers, and in these patients, relapse-freesurvival and overall survival are diminished (Slamon et al., Science,244:707-712 (1989); Slamon et al., Science, 235:177-182 (1987)).Alternatively, or additionally, FISH assays such as the INFORM™ (sold byVentana, Arizona) or PATHVISION™ (Vysis, Illinois) may be carried out onformalin-fixed, paraffin-embedded tumor tissue to determine the extent(if any) of HER2 overexpression in the tumor.

In one embodiment, the cancer will be one which expresses (and mayoverexpress) EGFR, such expression may be evaluated as for the methodsfor evaluating HER2 expression as noted above.

HER receptor or HER ligand overexpression or amplification may also beevaluated using an in vivo diagnostic assay, e.g. by administering amolecule (such as an antibody) which binds the molecule to be detectedand is tagged with a detectable label (e.g. a radioactive isotope) andexternally scanning the patient for localization of the label.

VI. Treatment with the HER2 Antibody Composition

It is contemplated that, according to the present invention, the HER2antibody may be used to treat cancer. The cancer will generally compriseHER2-expressing cells, such that the HER2 antibody herein is able tobind to the cancer cells. Various cancers that can be treated with thecomposition are listed in the definitions section above.

It is also contemplated that the HER2 antibody may be used to treatvarious non-malignant diseases or disorders, such as autoimmune disease(e.g. psoriasis); endometriosis; scleroderma; restenosis; polyps such ascolon polyps, nasal polyps or gastrointestinal polyps; fibroadenoma;respiratory disease; cholecystitis; neurofibromatosis; polycystic kidneydisease; inflammatory diseases; skin disorders including psoriasis anddermatitis; vascular disease; conditions involving abnormalproliferation of vascular epithelial cells; gastrointestinal ulcers;Menetrier's disease, secreting adenomas or protein loss syndrome; renaldisorders; angiogenic disorders; ocular disease such as age relatedmacular degeneration, presumed ocular histoplasmosis syndrome, retinalneovascularization from proliferative diabetic retinopathy, retinalvascularization, diabetic retinopathy, or age related maculardegeneration; bone associated pathologies such as osteoarthritis,rickets and osteoporosis; damage following a cerebral ischemic event;fibrotic or edemia diseases such as hepatic cirrhosis, lung fibrosis,carcoidosis, throiditis, hyperviscosity syndrome systemic, OslerWeber-Rendu disease, chronic occlusive pulmonary disease, or edemafollowing burns, trauma, radiation, stroke, hypoxia or ischemia;hypersensitivity reaction of the skin; diabetic retinopathy and diabeticnephropathy; Guillain-Barre syndrome; graft versus host disease ortransplant rejection; Paget's disease; bone or joint inflammation;photoaging (e.g. caused by UV radiation of human skin); benign prostatichypertrophy; certain microbial infections including microbial pathogensselected from adenovirus, hantaviruses, Borrelia burgdorferi, Yersiniaspp. and Bordetella pertussis; thrombus caused by platelet aggregation;reproductive conditions such as endometriosis, ovarian hyperstimulationsyndrome, preeclampsia, dysfunctional uterine bleeding, ormenometrorrhagia; synovitis; atheroma; acute and chronic nephropathies(including proliferative glomerulonephritis and diabetes-induced renaldisease); eczema; hypertrophic scar formation; endotoxic shock andfungal infection; familial adenomatosis polyposis; neurodegenerativediseases (e.g. Alzheimer's disease, AIDS-related dementia, Parkinson'sdisease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinalmuscular atrophy and cerebellar degeneration); myelodysplasticsyndromes; aplastic anemia; ischemic injury; fibrosis of the lung,kidney or liver; T-cell mediated hypersensitivity disease; infantilehypertrophic pyloric stenosis; urinary obstructive syndrome; psoriaticarthritis; and Hasimoto's thyroiditis. Preferred non-malignantindications for therapy herein include psoriasis, endometriosis,scleroderma, vascular disease (e.g. restenosis, artherosclerosis,coronary artery disease, or hypertension), colon polyps, fibroadenoma orrespiratory disease (e.g. asthma, chronic bronchitis, bronchieactasis orcystic fibrosis).

Treatment with the HER2 antibody will result in an improvement in thesigns or symptoms of disease. For instance, where the disease beingtreated is cancer, such therapy may result in an improvement in survival(overall survival and/or progression free survival) and/or may result inan objective clinical response (partial or complete).

Preferably, the HER2 antibody in the composition administered is a nakedantibody. However, the HER2 antibody administered may be conjugated witha cytotoxic agent. Preferably, the immunoconjugate and/or HER2 proteinto which it is bound is/are internalized by the cell, resulting inincreased therapeutic efficacy of the immunoconjugate in killing thecancer cell to which it binds. In a preferred embodiment, the cytotoxicagent targets or interferes with nucleic acid in the cancer cell.Examples of such cytotoxic agents include maytansinoids, calicheamicins,ribonucleases and DNA endonucleases.

The HER2 antibody is administered to a human patient in accord withknown methods, such as intravenous administration, e.g., as a bolus orby continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Intravenous administration of antibody composition is preferred.

For the prevention or treatment of disease, the appropriate dosage ofHER2 antibody will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether the HER2antibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theHER2 antibody, and the discretion of the attending physician. The HER2antibody is suitably administered to the patient at one time or over aseries of treatments. Depending on the type and severity of the disease,about 1 μg/kg to 50 mg/kg (e.g. 0.1-20 mg/kg) of HER2 antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. In one embodiment, the initial infusion time for the HER2antibody may be longer than subsequent infusion times, for instanceapproximately 90 minutes for the initial infusion, and approximately 30minutes for subsequent infusions (if the initial infusion is welltolerated). The preferred dosage of the HER2 antibody will be in therange from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more dosesof about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combinationthereof) may be administered to the patient. Such doses may beadministered intermittently, e.g. every week or every three weeks (e.g.such that the patient receives from about two to about twenty, e.g.about six doses of the HER2 antibody). An initial higher loading dose,followed by one or more lower doses may be administered. In oneembodiment, the HER2 antibody is administered as a loading dose ofapproximately 840 mg followed by approximately 420 mg approximatelyevery 3 weeks. In another embodiment, the HER2 antibody is administeredas a dose of approximately 1050 mg administered approximately every 3weeks.

Other therapeutic agents may be combined with the HER2 antibody. Suchcombined administration includes coadministration or concurrentadministration, using separate formulations or a single pharmaceuticalformulation, and consecutive administration in either order, whereinpreferably there is a time period while both (or all) active agentssimultaneously exert their biological activities. Thus, the othertherapeutic agent may be administered prior to, or following,administration of the HER2 antibody. In this embodiment, the timingbetween at least one administration of the other therapeutic agent andat least one administration of the HER2 antibody is preferablyapproximately 1 month or less, and most preferably approximately 2 weeksor less. Alternatively, the other therapeutic agent and the HER2antibody are administered concurrently to the patient, in a singleformulation or separate formulations.

Examples of other therapeutic agents that can be combined with the HER2antibody include any one or more of: a chemotherapeutic agent, such asan anti-metabolite, e.g. gemcitabine; a second, different HER2 antibody(for example, a growth inhibitory HER2 antibody such as Trastuzumab, ora HER2 antibody which induces apoptosis of a HER2-overexpressing cell,such as 7C2, 7F3 or humanized variants thereof); a second antibodydirected against another tumor associated antigen, such as EGFR, HER3,HER4; anti-hormonal compound, e.g., an anti-estrogen compound such astamoxifen, or an aromatase inhibitor; a cardioprotectant (to prevent orreduce any myocardial dysfunction associated with the therapy); acytokine; an EGFR-targeted drug (such as TARCEVA®, IRESSA® orCetuximab); an anti-angiogenic agent (especially Bevacizumab sold byGenentech under the trademark AVASTIN™); a tyrosine kinase inhibitor; aCOX inhibitor (for instance a COX-1 or COX-2 inhibitor); non-steroidalanti-inflammatory drug, Celecoxib (CELEBREX®); farnesyl transferaseinhibitor (for example, Tipifarnib/ZARNESTRA® R115777 available fromJohnson and Johnson or Lonafarnib SCH66336 available fromSchering-Plough); antibody that binds oncofetal protein CA 125 such asOregovomab (MoAb B43.13); HER2 vaccine (such as HER2 AutoVac vaccinefrom Pharmexia, or APC8024 protein vaccine from Dendreon, or HER2peptide vaccine from GSK/Corixa); another HER targeting therapy (e.g.trastuzumab, cetuximab, gefitinib, erlotinib, CI1033, GW2016 etc); Rafand/or ras inhibitor (see, for example, WO 2003/86467); Doxil;Topetecan; taxane; GW572016; TLK286; EMD-7200; a medicament that treatsnausea such as a serotonin antagonist, steroid, or benzodiazepine; amedicament that prevents or treats skin rash or standard acne therapies,including topical or oral antibiotic; a body temperature-reducingmedicament such as acetaminophen, diphenhydramine, or meperidine;hematopoietic growth factor, etc.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the agent and HER2 antibody. Treatment with the combination of theHER2 antibody composition and other therapeutic agent may result in asynergistic, or greater than additive, therapeutic benefit to thepatient.

If a chemotherapeutic agent is administered, it is usually administeredat dosages known therefor, or optionally lowered due to combined actionof the drugs or negative side effects attributable to administration ofthe chemotherapeutic agent. Preparation and dosing schedules for suchchemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,Baltimore, Md. (1992).

In addition to the above therapeutic regimes, the patient may besubjected to surgical removal of cancer cells and/or radiation therapy.

VII. Deposit of Materials

The following hybridoma cell lines have been deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, USA (ATCC):

Antibody Designation ATCC No. Deposit Date 7C2 ATCC HB-12215 Oct. 17,1996 7F3 ATCC HB-12216 Oct. 17, 1996 4D5 ATCC CRL 10463 May 24, 1990 2C4ATCC HB-12697 Apr. 8, 1999

Further details of the invention are illustrated by the followingnon-limiting Example. The disclosures of all citations in thespecification are expressly incorporated herein by reference.

Example Characterization of Pertuzumab Compositions

Pertuzumab is a recombinant humanized monoclonal antibody, generatedbased on human IgG1(κ) framework. It comprises two heavy chains (448residues) and two light chains (214 residues). The two heavy chains arelinked by two interchain disulfides and each light chain is attached toa heavy chain through one interchain disulfide. There is an N-linkedglycosylation site in the Fc region of pertuzumab at Asn-299 of the twoheavy chains. Pertuzumab differs from HERCEPTIN® (Trastuzumab) in theepitope binding regions of the light chain (12 amino acid differences)and the heavy chain (30 amino acid differences). As a result of thesedifferences, pertuzumab binds to a completely different epitope on theHER2 receptor. Binding of pertuzumab to the HER2 receptor on humanepithelial cells prevents it from forming complexes with other membersof the HER receptor family (Agus et al., Cancer Cell 2:127-137 (2002)).By blocking complex formation, pertuzumab prevents thegrowth-stimulatory effects of ligands for the complexes (e.g., EGF andheregulin). In vitro experiments demonstrated that both pertuzumab andpertuzumab-Fab inhibit the binding of heregulin (HRG) to MCF7 cells, andthat the HRG-stimulated phosphorylation of the HER2-HER3 complex can beinhibited by both pertuzumab and pertuzumab-Fab (Agus et al., CancerCell 2:127-137 (2002)). Furthermore, in vivo inhibition of tumor growthby pertuzumab and a polyethylene glycol derivatized Fab of pertuzumabwere found to be comparable in a murine prostate cancer xenograft model(Agus et al., Cancer Cell 2:127-137 (2002)). These data suggest that theFc region of the antibody is not necessary for the inhibition of tumorgrowth, and moreover, bivalency and Fc-mediated effector functions arenot required for in vivo or in vitro biological activity.

The following samples expressed by recombinantly engineered ChineseHamster Ovary (CHO) cells were analyzed:

Sample Manufacturing Process Scale Reference Material Phase I 400 L LotS9802A Phase II 2000 L  Process Development Materials Clinicaldevelopment program 400 L (Runs 1, 2, 3, 5, and 6) including Phase IIINote: 400 L Run 4 not available because of contamination at the 100 Linoculum culture at Day 2.N-Terminal Sequence Analysis

N-terminal sequence analysis was performed using standard Edmandegradation methods, and the results are shown in Table 2A, Table 2B,and Table 2C for the Reference Material, Lot S9802A, and the 400 L scaleRun 1 process development material, respectively. The expectedN-terminal sequences (FIG. 3A and FIG. 3B) of the light and heavy chainswere observed in all samples. An additional minor sequence correspondingto the light chain with three additional amino acids Val-His-Ser (VHS)preceding the expected N-terminal sequence was also detected in the fivesamples. The VHS sequence is a portion of the signal peptide that isremoved from protein as it is secreted. An alternate cleavage of thesignal peptide results in the VHS extension at the N-terminus ofpertuzumab. In the materials previously produced for Phase I/II clinicalstudies, about 2%-4% of the pertuzumab molecules have one of the twolight chains containing the N-terminal VHS sequence. However, the levelof light chains with N-terminal VHS sequence in these materials (1%-2%of each light chain) was too low to be detected in the N-terminalsequence analysis. In the five process development samples, the level ofthis light chain species is slightly above the detection limit of theN-terminal analysis at about 4%-5%. The N-terminal sequencing data forthe five process development samples are consistent with the cationexchange chromatographic results, which show that the five processdevelopment samples have approximately 9% of the pertuzumab moleculeswith one light chain containing the VHS extension (see Cation ExchangeChromatography (CEC) below).

No other sequences were detected (limit of detection estimated at 3%),indicating the absence of internal cleavage sites.

Mass Spectrometric Analysis

The Pertuzumab samples were reduced with dithiothreitol and analyzed byelectrospray-ionization mass spectrometry (ESI-MS) using a PE SCIEX API3000™ mass spectrometer to confirm that the masses of the heavy andlight chains are consistent with their expected sequences. Thereconstructed mass spectra for the Reference Material, Lot S9802A, andthe 400 L scale Run 1 process development material are compared in FIG.8A and FIG. 8B. The observed light chain mass (23,524 Da) is consistentwith the value predicted from its sequence for all three materials. Twoadditional minor peaks at 23,685 Da and 23,847 Da (161 and 323 Da higherthan the light chain mass, respectively) were observed. The first peak(23,685 Da) is likely to arise from glycation in the light chain. Thesecond peak (23,847 Da) is observed more clearly in the 400 L scaleprocess development materials. This peak is presumably from the lightchain with the Val-His-Ser extension (see the N-Terminal SequenceAnalysis above and the Cation Exchange Chromatographic Analysis) or thelight chain with two glycation sites.

Several masses corresponding to different forms of the heavy chain wereobserved. The predominant species of the heavy chain has a mass of50,532 Da, arising from the heavy chain consisting of residues 1-448with a G0 oligosaccharide structure. Other observed forms include theheavy chain containing residues 1-448 with a G1 or G2 oligosaccharidestructure (FIG. 8B);

Charge Heterogeneity by Cation Exchange Chromatography and CapillaryZone Electrophoresis

Cation exchange chromatography (CEC) was used to assess the chargeheterogeneity in Pertuzumab. Samples, before and after treatment withcarboxypeptidase B (CPB), were analyzed with a DIONEX™ cation exchangecolumn (PROPAC WCX-10™, 4 mm×250 mm) using a pH 6.0, 20 mM MES buffer,and shallow NaCl gradient. The comparison of chromatograms are shown inFIG. 9A (before CPB treatment) and FIG. 9B (after CPB treatment).Multiple peaks were observed in all three lots. For characterizationpurposes, the chromatograms are divided into six regions (labeled Athrough F in FIGS. 9A and 9B). Relative peak areas for the six regionsare listed in Table 3A and Table 3B. The amount of acidic variant (inregion A) is higher in Lot S9802A and the 400 L scale processdevelopment materials than in the Reference Material. The basic variantsin region C and D, which have been shown to contain Pertuzumab with aC-terminal lysine residue on one (region C) or both (region D) of theheavy chains, are reduced in Lot S9802A and the 400 L scale processdevelopment materials when compared to the Reference Material. After CPBtreatment, the Pertuzumab molecules with one or two heavy chainC-terminal lysine were converted to main species in region B and nolonger detected in region C and D. Only a small peak, whose identity hasnot yet been determined, remained in region C after CPB treatment. Thebasic variant in region E, shown to arise from the Pertuzumab moleculeswith one light chain containing the VHS extension, is higher in the 400L scale process development materials (9%-10%) than in the ReferenceMaterial and Lot S9802A (4%). The basic variant in region F was shown tobe Pertuzumab containing N-terminal VHS extension on one light chain anda C-terminal lysine on one heavy chain. As a result of higher levels ofacidic and basic variants, the main species in region B is lower in the400 L scale process development materials than in the Reference Materialor Lot S9802A.

In addition to cation exchange chromatography, capillary zoneelectrophoresis (CZE) was employed to examine the charge heterogeneityin Pertuzumab. The CZE peaks was identified and correlated to thosepeaks observed in CEC analysis by analyzing individually collected CECfractions with CZE. The relative amounts of charge variants determinedby CZE and CEC are comparable for all the materials analyzed.

The biological activities of Pertuzumab charge variants in different CECfractions appear to be the same based on a cell-based anti-proliferationassay. Thus, the charge heterogeneity in Pertuzumab is not expected toaffect its potency. The biological activities of the 400 L processdevelopment materials are comparable to those of the Reference Materialand Lot S9802A (see Biological Activity and Table 4).

Size-Exclusion Chromatography

Size-exclusion chromatography (SEC) was used to determine the extent ofaggregation in Pertuzumab. Samples were analyzed on a TOSOHAAS TSKG3000SWXL™ column (7.8 mm×300 mm). Isocratic elution was used (100 mMpotassium phosphate, pH 6.8, 0.5 mL/minutes) with ultraviolet (UV)absorbance monitored at 280 nm. SEC data for the Reference Material, LotS9802A, and the 400 L scale Run 1 process development material aredisplayed in FIG. 10. The monomer is consistently at greater than 99.6%by peak area, and the aggregate is below 0.3% in all materials analyzed.

CE-SDS-LIF Analysis

Capillary electrophoresis-sodium dodecyl sulfate analysis withlaser-induced fluorescence (CE-SDS-LIF) was also used to assess thepurity of Pertuzumab samples. Similar electropherograms were observedfor the Reference Material, Lot S9802A, and the 400 L scale Run 1process development material with and without reduction (FIGS. 11A and11B). The levels of non-glycosylated heavy chain determined from theelectropherograms of reduced materials are between 2.6% and 4.3% (withrespect to total heavy chain). The amounts of aggregates in thesesamples (non-reduced) determined by CE-SDS-LIF analysis are consistentwith the SEC results. No evidence of significant product fragments orother impurities were found in the CE-SDS-LIF analysis of these samples.The CE-SDS-LIF data suggest that the impurity profile of the 400 L scaleprocess development materials are similar to those of the ReferenceMaterial and Lot S9802A.

SDS-PAGE Analysis

The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (4%-20% Tgel (Daiichi Pure Chemicals Co., Tokyo, Japan) with SYPRORUBY™ staining)was performed to compare the reduced and intact samples of the ReferenceMaterial, Lot S9802A, and the 400 L scale process development materials.No new bands were observed in the 400 L scale process developmentmaterials, indicating again that the overall impurity profile in the 400L scale process development materials are similar to the ReferenceMaterial and Lot S9802A.

Tryptic and LYS-C Peptide Map Analyses

Tryptic and Lys-C peptide map analyses were performed to examine andcompare the primary structure of Pertuzumab in the Reference Material,Lot S9802A, and the 400 L scale process development materials. Aliquotsof reduced and S-carboxymethylated Pertuzumab were digested withtrypsin, and aliquots of reduced and sulfitolyzed Pertuzumab weredigested with endoproteinase Lys-C. The trypsin digest was separated byreversed-phase chromatography using a VYDAC C-18™ column (4.6 mm×250 mm)with a 0%-60% acetonitrile gradient. Absorbance was monitored at 214 nm,and peptide masses were determined by ESI-MS using a THERMO FINNIGANLCQ™. The Lys-C digest was separated by reversed-phase chromatographyusing a ZORBAX C-8™ column (4.6 mm×150 mm) with a 0%-100% isopropylalcohol (IPA) gradient. Absorbance was monitored at 214 nm, and peptidemasses were determined by ESI-MS using a THERMO FINNIGAN LCQ™.

The tryptic and Lys-C peptide maps for the Reference Material, LotS9802A, and the 400 L scale Run 1 process development material arecompared in FIG. 12A and FIG. 12B, respectively. Both tryptic and Lys-Cmaps for all three materials are essentially identical. The majority ofthe peptides were identified by LC-MS and matched to an expected peptidemass. The observed tryptic peptides identified 97.1% (436/449) of theheavy chain residues and 95.8% (205/214) of the light chain residues.The sequence coverage is 98.4% (442/449) and 70.6% (151/214) for theheavy and light chain, respectively, in the Lys-C map. The N-terminalpeptide containing the VHS extension was found to co-elute with theN-terminal peptide without the VHS extension in the tryptic peptide mapby LC-MS. No significant amounts of deamidated or oxidized peptides weredetected in the peptide maps.

Biological Activity

The biological activity of Pertuzumab was determined by measuring itsability to inhibit proliferation of the human breast cancer cell lineMDA-MB-175-VII. The percent specific activities obtained for five 400 Lscale process development samples (Table 4) are in the range of 90%-96%and similar to the activities for the Reference Material (100% bydefinition) and Lot S9802A (98% reported in Certificate of Analysis). Asexpected, the charge heterogeneity does not affect the potency ofPertuzumab. All materials have comparable anti-proliferation activities.

N-Linked Oligosaccharide Analysis

Part of the mass heterogeneity in the heavy chain arises from theglycosylation at Asn-299. To assess the heterogeneity of theoligosaccharides, Pertuzumab samples were digested overnight with PNGaseF to release the N-linked glycans. The released oligosaccharides werethen derivatized with the fluorophore 9-amino-1,4,6-trisulfonate (APTS).Individual glycan forms were separated by capillary electrophoresis (CE)(Beckman P/ACE 5500 CE equipped with Beckman coated capillary) andquantified with fluorescence detection.

The electropherograms from CE analysis of the released neutraloligosaccharides from the Reference Material, Lot S9802A, and the 400 Lscale Run 1 process development material are shown in FIG. 13. Forreference, the oligosaccharide structures commonly found in human IgG1antibodies, and a summary of the nomenclature used are included in FIG.14. Relative amounts of oligosaccharides in all materials are summarizedin Table 5.

The oligosaccharides with G0 and G1 structures are the predominantglycans. Peaks arising from other oligosaccharide structures were alsoobserved in the electropherograms. These structures include G2, G0-F,G-1, Man5, and G1-1 (or Man6) glycoforms. In addition, the isoforms areresolved. The distributions of observed glycans are similar in allmaterials (Table 5). However, compared to the Reference Materials and400 L scale process development materials, the Phase II material (LotS9802A) has a smaller amount of glycans with a G0 structure and moreglycans with G1 structure. Despite the differences in glycandistribution, all materials have similar biological activities. Inaddition, the change in glycan heterogeneity did not have a significantimpact on the binding affinity of Pertuzumab for FcRn (Table 6) or Fcgamma receptors (Table 7) (see FcRn Receptor and Fc Gamma ReceptorBinding Assays).

The released neutral oligosaccharides were also analyzed by MALDI-TOFmass spectrometry (MALDI-TOF/MS). The MALDI-TOF spectra for the releasedneutral oligosaccharides from the Reference Material, Lot S9802A, and400 L scale Run 1 process development material are compared in FIG. 15.The glycan structure and distribution obtained from MALDI-TOF/MSanalysis are consistent with the CE results.

Capillary Isoelectric Focusing Analysis

Capillary isoelectric focusing (cIEF) was used to determine the pI ofPertuzumab in the Reference Material, Lot S9802A, and the 400 L scaleRun 1 process development material. Although the relative amounts of thedifferent charged species in these materials were somewhat different asobserved in the CEC analysis, the pI of the main species was found to be8.7 in all samples.

Free Sulfhydryl Analysis

The Reference Material, Lot S9802A, and the 400 L scale processdevelopment materials were tested for free thiol (unpaired cysteineresidue) using the Ellman's analysis. Free thiol level was below thelimit of detection (approximately 0.05 mole free thiol per mole protein)in all materials tested.

FcRn Receptor Binding Assay

The FcRn receptor binding affinities of Pertuzumab from the ReferenceMaterial, Lot S9802A, and the 400 L scale process development materialswere compared using an ELISA assay similar to the one described inShields et al., J. Biol. Chem. 276:6591-6604 (2001). The ReferenceMaterial was used as a standard in this assay.

MAXISORP™ 96-well microwell plates (Nunc, Roskilde, Denmark) were firstcoated with NEUTRAVADIN™ (Pierce, Rockford, Ill.) at 4° C. overnight.Biotinylated human FcRn was then added to the plates at 2 μg/mL andincubated for one hour. Eleven two-fold serial dilutions of Pertuzumabsamples (3.1-3200 ng/mL) were added to the plates and incubated for twohours. Bound Pertuzumab was detected by adding peroxidase labeled goatF(ab′)₂ anti-human IgG F(ab′)₂ (Jackson ImmunoResearch, West Grove, Pa.)and using 3,3′,5,5′-tetramethyl benzidine (TMB) (Kirkegaard & PerryLaboratories, Gaithersburg, Md.) as the substrate. Absorbance was readat 450 nm on a TITERTEK MULTISKAN™ (MCC/340) reader (ICN, Costa Mesa,Calif.). The FcRn binding affinity of Pertuzumab was also evaluated in asecond ELISA format in which HER2-ECD was coated on plates. Seriallydiluted Pertuzumab samples were added to the plates and incubated fortwo hours. Plates were washed and 2 mg/mL of biotinylated human FcRnwere added. Bound FcRn was detected using streptavidin-HRP with TMB asthe substrate.

The absorbance at the midpoint of the titration curve (mid-OD) ofstandard (i.e., Reference Material) and the corresponding concentrationsof standard and samples at this mid-OD were determined. The relativebinding affinity was calculated by dividing the mid-OD concentration ofstandard by that of the sample.

The relative FcRn binding affinities obtained from both ELISA formatsare listed in Table 6. Data from the two different ELISA formats arecomparable. Although the glycan distributions in different Pertuzumabmaterials are not identical, the FcRn binding affinities of thesematerials are comparable, indicating that the glycan heterogeneity inPertuzumab has no apparent effect on its FcRn binding affinity.

Fc Gamma Receptor Binding Assay

Binding of Pertuzumab to the human Fc gamma receptors (FcγR) wasassessed by an ELISA assay according to Shields et al., J. Biol. Chem.276:6591-6604 (2001) with modifications.

Monomeric IgG can bind to the high-affinity FcγRIa (CD64); however, thelow-affinity receptors (FcγRIIa (CD32A), FcγRIIb (CD32B), and FcγRIIIa(CD16)) require multimeric IgG for significant binding. Therefore, forthe low-affinity receptor binding assays, multimers of Pertuzumab wereformed before assay by mixing each sample (200 mg/mL) with goatanti-human kappa chain (400 mg/mL; ICN Biomedical, Irvine, Calif.). Thehuman FcγR were expressed as recombinant fusion proteins of theextracellular domain of the receptor alpha chains with Gly/His6/GST(glycine/6 histidines/glutathione-s-transferase) Anti-GST-coated, bovineserum albumin (BSA)-blocked assay plates were used to capture the FcγR.The receptors (100 mL at 0.25 mg/mL) were added to the plates andincubated for 1 hour. Serial dilutions of Pertuzumab samples (100 mL)were added as monomers for FcγRIa and as multimers for the low-affinityFcγR, and the plates were incubated for two hours. The bound Pertuzumabwas detected by adding horseradish peroxidase-conjugated goat anti-humanF(ab′)₂ (Jackson ImmunoResearch Laboratories, West Grove, Pa.) and usingTMB as substrate. EC₅₀ values for binding of Pertuzumab to the FcγR weredetermined by nonlinear regression analysis with a four-parameter model(KaleidaGraph, Synergy Software, Reading, Pa.). RITUXAN® (LotC2B81298-2) was used as the control antibody.

The EC₅₀ values for binding of Pertuzumab to FcγR are summarized inTable 7. The results show that the Reference Material, Lot S9802A, andthe 400 L scale process development materials have comparable bindingaffinities for FcγR. These results suggest that the glycan heterogeneityin Pertuzumab has no significant effect on its FcγR-binding affinities.

TABLE 2A N-Terminal Sequence Analysis of Pertuzumab Reference Material(nmol of residue observed in each cycle) Cycle Residue 1 2 3 4 5 6 7 8 910 11 12 ALA 0.01 0.01 0.02 0.04 0.04 0.05 0.06 0.07 0.09 0.09 0.10 0.11ARG 0.01 0.01 0.01 0.01 0.03 0.03 0.04 0.04 0.05 0.06 0.05 0.06 ASN 0.000.00 0.01 0.01 0.01 0.02 0.03 0.04 0.05 0.06 0.06 0.07 ASP 0.48 0.080.03 0.04 0.04 0.04 0.05 0.07 0.07 0.08 0.09 0.10 CYS NA NA NA NA NA NANA NA NA NA NA NA GLN 0.00 0.01 0.92 0.13 0.04 0.46 0.13 0.07 0.08 0.080.08 0.09 GLU 0.49 0.05 0.13 0.03 0.03 0.51 0.12 0.07 0.07 0.07 0.070.08 GLY 0.01 0.01 0.03 0.03 0.05 0.05 0.05 0.30 0.35 0.36 0.13 0.09 HIS0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 ILE 0.000.48 0.06 0.02 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.04 LEU 0.00 0.030.04 0.43 0.11 0.08 0.09 0.11 0.12 0.13 0.62 0.26 LYS 0.00 0.00 0.030.00 0.05 0.06 0.07 0.08 0.08 0.09 0.00 0.12 MET 0.00 0.00 0.00 0.530.07 0.02 0.02 0.02 0.02 0.02 0.02 0.02 PHE 0.00 0.01 0.02 0.02 0.030.04 0.05 0.05 0.06 0.07 0.07 0.08 PRO 0.00 0.01 0.02 0.03 0.04 0.040.05 0.32 0.13 0.09 0.09 0.10 SER 0.01 0.01 0.03 0.04 0.05 0.07 0.560.16 0.30 0.33 0.18 0.28 THR 0.00 0.01 0.00 0.04 0.33 0.12 0.07 0.070.08 0.09 0.10 0.10 TRP 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.010.02 0.02 0.02 TYR 0.01 0.01 0.03 0.04 0.05 0.06 0.08 0.08 0.09 0.100.11 0.13 VAL 0.01 0.51 0.08 0.10 0.53 0.15 0.11 0.12 0.14 0.15 0.160.51 Light Chain ASP ILE GLN MET THR GLN SER PRO SER SER LEU SER HeavyChain GLU VAL GLN LEU VAL GLU SER GLY GLY GLY LEU VAL Note: Thenanomoles of phenylthiohydantoin amino acid residues observed in eachcycle is given. Residues from the heavy chain are given in bold type,and residues from the light chain are underlined. Approximately 0.5 nmolof protein was loaded, equivalent to 1.0 nmol of each of the light andheavy chains. Cysteine (CYS) is not observed in the sequence analysis.

TABLE 2B N-Terminal Sequence Analysis of Pertuzumab Lot S9802A (nmol ofresidue observed in each cycle) Cycle Residue 1 2 3 4 5 6 7 8 9 10 11 12ALA 0.01 0.01 0.03 0.04 0.06 0.06 0.08 0.09 0.11 0.12 0.14 0.14 ARG 0.010.01 0.02 0.02 0.03 0.03 0.05 0.05 0.06 0.07 0.07 0.08 ASN 0.00 0.000.01 0.02 0.02 0.02 0.04 0.05 0.06 0.07 0.08 0.09 ASP 0.61 0.12 0.040.05 0.05 0.06 0.07 0.09 0.09 0.10 0.11 0.13 CYS NA NA NA NA NA NA NA NANA NA NA NA GLN 0.00 0.01 1.11 0.22 0.06 0.55 0.19 0.10 0.11 0.11 0.110.12 GLU 0.61 0.09 0.15 0.05 0.04 0.61 0.19 0.10 0.09 0.10 0.10 0.11 GLY0.01 0.01 0.04 0.04 0.06 0.06 0.07 0.37 0.44 0.46 0.19 0.13 HIS 0.000.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 ILE 0.00 0.560.10 0.02 0.04 0.04 0.03 0.04 0.05 0.05 0.05 0.06 LEU 0.00 0.04 0.050.51 0.17 0.11 0.12 0.14 0.15 0.17 0.76 0.37 LYS 0.00 0.00 0.04 0.000.06 0.08 0.09 0.10 0.11 0.12 0.00 0.16 MET 0.00 0.00 0.01 0.61 0.120.03 0.03 0.03 0.02 0.02 0.02 0.03 PHE 0.00 0.01 0.03 0.03 0.04 0.050.06 0.07 0.08 0.09 0.09 0.10 PRO 0.00 0.01 0.02 0.03 0.05 0.06 0.060.37 0.18 0.12 0.12 0.13 SER 0.01 0.02 0.04 0.05 0.06 0.08 0.63 0.240.36 0.41 0.24 0.35 THR 0.00 0.01 0.00 0.05 0.39 0.17 0.10 0.10 0.110.12 0.13 0.14 TRP 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.02 0.02 0.020.02 0.02 TYR 0.16 0.07 0.05 0.06 0.07 0.08 0.10 0.10 0.12 0.13 0.140.16 VAL 0.02 0.58 0.13 0.12 0.62 0.22 0.15 0.16 0.18 0.20 0.21 0.62Light Chain ASP ILE GLN MET THR GLN SER PRO SER SER LEU SER Heavy ChainGLU VAL GLN LEU VAL GLU SER GLY GLY GLY LEU VAL Note: The nanomoles ofphenylthiohydantoin amino acid residues observed in each cycle is given.Residues from the heavy chain are given in bold type, and residues fromthe light chain are underlined. Approximately 0.5 nmol of protein wasloaded, equivalent to 1.0 nmol of each of the light and heavy chains.Cysteine (CYS) is not observed in the sequence analysis.

TABLE 2C N-Terminal Sequence Analysis of Pertuzumab 400 L Scale Run 1(nmol of residue observed in each cycle) Cycle Residue 1 2 3 4 5 6 7 8 910 11 12 ALA 0.01 0.02 0.03 0.05 0.06 0.06 0.08 0.10 0.11 0.13 0.14 0.14ARG 0.03 0.05 0.07 0.09 0.17 0.15 0.20 0.23 0.24 0.29 0.30 0.33 ASN 0.000.00 0.01 0.02 0.02 0.03 0.04 0.06 0.07 0.08 0.09 0.09 ASP 0.89 0.080.05 0.11 0.08 0.09 0.11 0.15 0.14 0.16 0.18 0.19 CYS NA NA NA NA NA NANA NA NA NA NA NA GLN 0.00 0.01 1.17 0.11 0.05 0.60 0.12 0.09 0.14 0.120.11 0.12 GLU 0.68 0.05 0.19 0.04 0.04 0.62 0.15 0.10 0.10 0.11 0.110.12 GLY 0.01 0.01 0.05 0.04 0.08 0.07 0.07 0.37 0.42 0.44 0.16 0.13 HIS0.00 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.03 0.03 0.03 ILE 0.000.56 0.03 0.02 0.07 0.04 0.03 0.04 0.05 0.06 0.05 0.06 LEU 0.00 0.040.05 0.51 0.15 0.12 0.13 0.15 0.16 0.18 0.79 0.31 LYS 0.00 0.00 0.040.05 0.08 0.09 0.10 0.12 0.14 0.15 0.17 0.19 MET 0.00 0.00 0.01 0.650.04 0.02 0.05 0.03 0.02 0.02 0.03 0.03 PHE 0.00 0.01 0.03 0.03 0.050.05 0.07 0.08 0.08 0.09 0.10 0.11 PRO 0.00 0.01 0.02 0.04 0.05 0.060.07 0.41 0.14 0.11 0.13 0.14 SER 0.01 0.02 0.07 0.06 0.08 0.10 0.760.21 0.43

0.23 0.42 THR 0.00 0.01 0.00 0.06 0.57 0.18 0.12 0.16 0.16 0.17 0.180.19 TRP 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.02 0.02 0.02 0.03 0.03 TYR0.00 0.02 0.04 0.06 0.07 0.08 0.10 0.11 0.13 0.14 0.15 0.17 VAL 0.040.56 0.10 0.13 0.62 0.19 0.15 0.17 0.19 0.21 0.22 0.62 Light Chain ASPILE GLN MET THR GLN SER PRO SER SER LEU SER Heavy Chain GLU VAL GLN LEUVAL GLU SER GLY GLY GLY LEU VAL VHS-Light Chain VAL HIS SER ASP ILE GLNMET THR GLN SER PRO SER Note: The nanomoles of phenylthiohydantoin aminoacid residues observed in each cycle is given. Residues from the heavychain are given in bold type, and residues from the light chain areunderlined. Residues from the additional VHS-light chain sequence areshown in italics. Approximately 0.5 nmol of protein was loaded,equivalent to 1.0 nmol of each of the light and heavy chains. Cysteine(CYS) is not observed in the sequence analysis. 0

TABLE 3A Cation Exchange Chromatographic Analysis of Native Pertuzumab(% peak area) Ion-Exchange Variant Peak Sample A B C D E F ReferenceMaterial 10 73 10 3 3 0.4 S9802A 20 68 5 2 4 0.2 400 L Scale Run 1 16 655 2 10 0.9 400 L Scale Run 2 15 67 5 2 9 0.6 400 L Scale Run 3 15 67 6 210 0.6 400 L Scale Run 5 15 67 5 2 9 0.5 400 L Scale Run 6 13 63 9 3 90.6 Note: 400 L Run 4 not available because of contamination of the 100L inoculum culture at Day 2.

TABLE 3B Cation Exchange Chromatographic Analysis of CPB-DigestedPertuzumab (% peak area) Ion-Exchange Variant Peak Sample A B C D E FReference Material 11 78 4 ND 4 ND S9802A 20 71 3 ND 4 ND 400 L ScaleRun 1 17 68 3 ND 10 ND 400 L Scale Run 2 15 71 3 ND 9 ND 400 L Scale Run3 15 71 3 ND 10 ND 400 L Scale Run 5 17 70 3 ND 9 ND 400 L Scale Run 616 71 3 ND 9 ND Note: Fractions A through F are defined in FIG. 9B. Allvalues are rounded to two significant figures. Totals in any row may notadd to 100% because of rounding. 400 L Run 4 not available because ofcontamination of the 100 L inoculum culture at Day 2. ND = Not detectedor cannot be integrated.

TABLE 4 Specific Activities of Pertuzumab by a Cell-BasedAnti-Proliferation Assay Material Specific Activity (%) % CV ReferenceMaterial 100^(a)  — S9802A 98^(b) 10 400 L Scale Run 1 96^(c) 18 400 LScale Run 2 90^(c) 11 400 L Scale Run 3 96^(c) 17 400 L Scale Run 596^(c) 3 400 L Scale Run 6 95^(c) 3 Note: 400 L Run 4 not availablebecause of contamination of the 100 L inoculum culture at Day 2. ^(a)Bydefinition, specific activity of Reference Material is 100%. ^(b)Valuereported for Lot S9802A. ^(c)Value represents mean of three assays.

TABLE 5 Distribution of Oligosaccharide Structures in PertuzumabDetermined by CE % Man6 + Sample % G0-F % G-1 % Man5 % G0 % G1-1 %G1^(a) % G2 Reference Material 1 4 1 71 2 19 2 S9802A 1 4 1 62 2 27 3400 L Scale Run 1 2 6 1 73 3 15 2 400 L Scale Run 2 1 6 1 77 2 13 1 400L Scale Run 3 2 6 1 74 4 14 1 400 L Scale Run 5 1 5 1 71 2 18 1 400 LScale Run 6 1 5 1 71 3 18 1 Note: 400 L Run 4 not available because ofcontamination of the 100 L inoculum culture at Day 2. ^(a)Sum of the twoG1 isomers.

TABLE 6 Relative Binding Affinities of Pertuzumab for FcRn RelativeBinding Affinity^(a) NeutrAvidin Her2ECD Sample Coat Format Coat FormatReference Material (Standard) 1.00 1.00 S9802A 1.34 1.30 400 L Scale Run1 1.04 1.22 400 L Scale Run 2 1.09 1.31 400 L Scale Run 3 1.14 1.42 400L Scale Run 5 1.22 1.36 400 L Scale Run 6 1.06 1.29 Note: 400 L Run 4not available because of contamination of the 100 L inoculum culture atDay 2. ^(a)The absorbance at the midpoint of the titration curve(mid-OD) of standard (i.e., Reference Material) and the correspondingconcentrations of standard and samples at this mid-OD were determined.The relative binding affinity was obtained by dividing the mid-ODconcentration of standard by that of the sample.

TABLE 7 EC₅₀ Values for Binding of Pertuzumab to Fc Gamma Receptors EC₅₀(μg/mL) FcγRIIIa FcγRIIIa FcγRIa FcγRIIa FcγRIIb (F158) (V158) SampleMean SD Mean SD Mean SD Mean SD Mean SD Reference Material 0.0081 0.00145.8 1.8 57 17 14.0 1.4 1.8 0.1 S9802A 0.0078 0.0020 5.4 1.9 45 17 7.80.3 1.1 0.1 400 L Scale Run 1 0.0079 0.0013 5.4 1.5 70 25 8.4 0.9 1.20.1 400 L Scale Run 2 0.0074 0.0016 5.8 1.6 54 18 10.4 0.7 1.4 0.2 400 LScale Run 3 0.0075 0.0015 5.5 1.1 54 10 8.2 0.9 1.2 0.1 400 L Scale Run5 0.0076 0.0011 6.1 2.7 50 12 10.9 1.5 1.4 0.2 400 L Scale Run 6 0.00800.0005 6.2 1.9 44 1 10.3 0.9 1.4 0.2 Note: The means and standarddeviations (SD) were obtained from multiple runs of the assay (N = 4).FcγRIIIa receptor has two versions: F158 and V158. 400 L Run 4 notavailable because of contamination of the 100 L inoculum culture at Day2.

1. A polypeptide comprising the amino acid sequence in SEQ ID No. 23, ora deamidated and/or oxidized variant thereof.
 2. An antibody comprising(a) a light chain comprising the polypeptide of claim 1, and (b) a heavychain comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO. 16, SEQ ID NO. 24, and a deamidated and/oroxidized variant of SEQ ID NO. 16 or SEQ ID NO.
 24. 3. A pharmaceuticalformulation comprising the antibody of claim 2 in a pharmaceuticallyacceptable carrier.
 4. An isolated antibody comprising (a) a light chaincomprising the polypeptide of claim 1, and (b) a heavy chain comprisingthe amino acid sequence selected from the group consisting of SEQ ID NO.16, SEQ ID NO. 24, and a deamidated and/or oxidized variant of SEQ IDNO. 16 or SEQ ID NO.
 24. 5. A pharmaceutical formulation comprising theantibody of claim 4 in a pharmaceutically acceptable carrier.
 6. Anisolated polypeptide comprising the amino acid sequence in SEQ ID No.23, or a deamidated and/or oxidized variant thereof.